Compositions for the treatment of diabetes

ABSTRACT

A supramolecular insulin assembly and supramolecular exendin-4 assembly, which is useful as a protein therapeutic agent for the treatment of metabolic disorders particularly diabetes. The supramolecular assemblies disclosed in the present invention consists of insoluble and aggregated oligomers the protein. The invention also provides pharmaceutical compositions comprising the supramolecular assembly.

RELATED APPLICATIONS

This application claims priority from Indian Patent Application No.:914/DEL/2008 filed on Apr. 7, 2008.

TECHNICAL FIELD

The present invention relates to protein therapeutics for treatment ofdiabetes and other chronic diseases.

BACKGROUND OF INVENTION

Protein medications are the most rapidly expanding class oftherapeutics, serving patients with diabetes, cancer, cardiovascular,renal, gastrointestinal, rheumatologic and neurological diseases, amongmany others. The therapeutic and commercial value of proteins astherapeutics including insulin, erythropoietin, G-CSF, plasminogenactivator, and interferons is undisputed. Improved proteins or theirformulations have enhanced the therapeutic efficacy of these parentproducts, by increasing their potency, time of action, and otherproperties.

Diabetes is a chronic disease characterized by either the inability ofthe body to produce insulin (Type I) or the failure to respond to it(Type II) (http://www.who.int/diabetes/en, King, H., Aubert, R. E. &Herman, W. H. Global burden of diabetes, 1995-2025: Prevalence,numerical estimates, and projections. Diabetes Care 21, 1414-1431(1998)). There is an emerging global epidemic of diabetes of both Type Iand Type II forms. Nearly 1.1 million diabetics succumbed to death in2005 (Dunstan, D. W., Zimmet, P. Z., Welborn, T. A., De Courten, M. P.,Cameron, A. J., et al. The rising prevalence of diabetes and impairedglucose tolerance: The Australian Diabetes, Obesity and Lifestyle Study.Diabetes Care 25, 829-834 (2002)). Its economic consequences are evenmore staggering as people may live for years with diabetes, their causeof death is often recorded as heart diseases and kidney failures, botharising as a secondary consequence of diabetes. Restoring the normalmetabolic milieu by administration of insulin from outside and therebyminimizing the risk of secondary complications has become an essentialfeature of diabetic treatment. The current therapy involves multipledaily subcutaneous (SC)/intramuscular (IM) injections of insulin, whichleads to a heavy burden of compliance on patients. This in turn has ledto alternative, less invasive routes of delivery. Attempts to exploitthe nasal, oral, gastrointestinal and transdermal routes, have beenmostly unsuccessful. Although a conventional insulin regimen for type Idiabetes with twice-daily insulin injections is effective in controllingpostprandial blood glucose levels, this treatment is of limited valuedue to its failure to control fasting hyperglycemia. Patients withdiabetes mellitus need insulin therapy to boost intrinsic insulin supplyonce or twice a day. Also, post-prandial glucose homeostasis ismaintained through regular insulin injections before each meal.Intensive insulin therapy delays the onset and or slows the progressionof secondary complications, yet patients remain at a high risk offasting hypoglycemia. An insulin formulation which releases insulin in acontrolled manner for long periods of time would free the patients fromthe need to administer multiple doses of insulin daily.

SUMMARY OF THE INVENTION

The invention provides a composition for a prolonged release of insulinand/or exendin-4 for treating diabetes, both type I and II, e.g.,protein therapeutics for treatment of diabetes and other chronicdiseases. Disclosed herein is a supramolecular insulin assembly, whereinthe supramolecular insulin assembly comprises the insoluble andaggregated oligomeric form of insulin and/or exendin-4. The presentinvention also discloses the supramolecular exendin-4 assembly (SEA).

One aspect of the present invention relates to a supramolecular insulinassembly (SIA), which is useful as a protein therapeutic for thetreatment of metabolic disorders selected from the group consisting oftype 1, type 2 diabetes mellitus and complications. The assemblycomprises insoluble and aggregated oligomeric form of insulin.

Another aspect of the present invention relates to a supramolecularinsulin assembly (SIA) useful as protein therapeutics for the treatmentof metabolic disorders selected from the group consisting of type 1,type 2 diabetes mellitus and complications thereof, wherein the assemblycomprises of insoluble and aggregated oligomeric form of insulin as SIAI, SIA II, SIA III or combination thereof. For example SIA I consists ofelongated clusters having pearl like arrangement of insulin monomer, SIAII consists of linear association of elongated clusters having pearllike arrangement of insulin monomer, or SIA III consists of about 90%SIA-II, that is a dense, linear association of insulin oligomers.

Another aspect of the present invention relates to a supramolecularinsulin assembly (SIA) useful as a protein therapeutic for the treatmentof metabolic disorders selected from the group consisting of type 1,type 2 diabetes mellitus and complications thereof, wherein the assemblycomprises insoluble and aggregated oligomeric form of insulin as SIA I,wherein SIA I consists of elongated clusters having pearl likearrangement of insulin monomer.

Yet another aspect of the present invention relates to a supramolecularinsulin assembly (SIA) useful as a protein therapeutic for the treatmentof metabolic disorders selected from the group consisting of type 1,type 2 diabetes mellitus and complications thereof, wherein the assemblycomprises insoluble and aggregated oligomeric form of insulin as SIA II,wherein SIA II consists of linear association of elongated clustershaving pearl like arrangement of insulin monomer.

Still another aspect of the present invention relates to asupramolecular insulin assembly (SIA) useful as a protein therapeuticfor the treatment of metabolic disorders selected from the groupconsisting of type 1, type 2 diabetes mellitus and complicationsthereof, wherein the assembly comprises insoluble and aggregatedoligomeric form of insulin as SIA III, wherein SIA III consists dense,linear association of insulin oligomers.

Further aspect of the present invention relates to a supramolecularexendin-4 assembly (SEA) useful as a protein therapeutic for thetreatment of diabetes, wherein said assembly comprises insoluble andaggregated oligomeric form of exendin-4.

Another aspect of the present invention relates to a pharmaceuticalcomposition for the treatment of metabolic disorders selected from thegroup consisting of type 1, type 2 diabetes mellitus and complicationsthereof, the composition comprising therapeutically effective amount ofthe supramolecular insulin assembly (SIA) as disclosed in the presentinvention.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition for the treatment of metabolic disorders selected from thegroup consisting of type 1, type 2 diabetes mellitus and complicationsthereof. The composition comprises therapeutically effective amount ofthe supramolecular insulin assembly-II (SIA-II).

Still another aspect of the present invention relates to apharmaceutical composition for the treatment of metabolic disordersselected from the group consisting of type 1, type 2 diabetes mellitusand complications thereof. The composition comprises a therapeuticallyeffective amount of the supramolecular insulin assembly (SIA) andsupramolecular exendin-4 assembly (SEA).

Still yet another aspect of the present invention relates to apharmaceutical composition for the treatment of metabolic disordersselected from the group consisting of type 1, type 2 diabetes mellitusand complications thereof, the composition comprising therapeuticallyeffective amount of the supramolecular insulin assembly (SIA) andsupramolecular exendin-4 assembly (SEA).

Yet another aspect of the present invention relates to a pharmaceuticalcomposition for the treatment of metabolic disorders selected from thegroup consisting of type 1, type 2 diabetes mellitus and complicationsthereof, the composition comprising therapeutically effective amount ofsupramolecular exendin-4 assembly (SEA).

Another aspect of the present invention relates to a process ofpreparation of supramolecular insulin assembly (SIA) as disclosed in thepresent invention, the process comprising; dissolving insulin at atemperature of about 25 to 60° C. in a solution having pH in the rangeof about 1.5 to 7.8; and incubating the above for a period of about 6 to48 hours with constant shaking to obtain Supramolecular Insulin Assembly(SIA), wherein SIA comprises insoluble and aggregated oligomeric form ofinsulin.

Yet another aspect of the present invention relates to a process ofpreparation of supramolecular exendin-4 assembly. The process comprisesdissolving exendin-4 at a temperature of about 25° C. to 60° C. in asolution having pH in the range of about 2.0 to 7.6; and incubating theabove for a period of 6 to 192 hours with constant shaking to obtainSupramolecular Exendin-4 Assembly (SEA), wherein SEA comprises insolubleand aggregated oligomeric form of Exendin-4.

Further aspect of the present invention relates to a method for treatingmetabolic disorders selected from the group consisting of type 1, type 2diabetes mellitus and complications thereof, wherein the methodcomprises administering to a subject in need thereof a therapeuticallyeffective amount of the pharmaceutical composition comprising thesupramolecular insulin assembly at a dose which is effective for thealleviation of the disorder.

Yet another aspect of the present invention relates to a method fortreating metabolic disorders selected from the group consisting of type1, type 2 diabetes mellitus and complications thereof, wherein themethod comprises administering to a subject in need thereof atherapeutically effective amount of the pharmaceutical compositioncomprising the supramolecular insulin assembly-II (SIA-II), which iseffective for the alleviation of the disorder.

Yet another aspect of the present invention relates to a method fortreating metabolic disorders selected from the group consisting of type1, type 2 diabetes mellitus and complications thereof, wherein themethod comprising administering to a subject in need thereof atherapeutically effective amount of the pharmaceutical compositioncomprising the supramolecular exendin-4 assembly, which is effective forthe alleviation of said disorder.

Still yet another aspect of the present invention relates to a methodfor treating metabolic disorders selected from the group consisting oftype 1, type 2 diabetes mellitus and complications thereof, wherein themethod comprising administering to a subject in need thereof atherapeutically effective amount of the pharmaceutical compositioncomprising the supramolecular insulin assembly and supramolecularexendin-4 assembly, which is effective for the alleviation of saiddisorder.

Another aspect of the present invention relates to use of supramolecularinsulin assembly for the treatment of metabolic disorders selected fromthe group consisting of type 1, type 2 diabetes mellitus andcomplications thereof.

Still yet another aspect of the present invention relates to use ofsupramolecular exendin-4 assembly for the treatment of metabolicdisorders selected from the group consisting of type 1, type 2 diabetesmellitus and complications thereof.

Still yet another embodiment of the present invention relates to use ofsupramolecular insulin assembly in combination with supramolecularexendin-4 assembly for the treatment of metabolic disorders selectedfrom the group consisting of type 1, type 2 diabetes mellitus andcomplications thereof.

Polypeptides or other compounds described herein are purified orisolated. A purified or isolated composition (e.g., protein,polypeptide) is at least 60% by weight (dry weight) the compound ofinterest. Preferably, the preparation is at least 75%, more preferablyat least 90%, and most preferably at least 99%, by weight the compoundof interest. Purity is measured by any appropriate standard method, forexample, by column chromatography, polyacrylaminde gel electrophoresis,or HPLC analysis. The polypeptide is purified from MSC culture media orrecombinantly produced.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph showing kinetics of fibril formation at pH 2.0and 7.0 monitored with 50 μM Th-T fluorescence of recombinant human (rH)and bovine insulin.

FIG. 2 a is a line graph showing in-vitro release of insulin fromsupramolecular insulin assembly II (also referred to as pre-amyloidinsulin II intermediate) of bovine and rH insulin monitored byabsorbance at 280 nm and intrinsic tyrosine fluorescence. The Th-Tintensity of solution inside the dialysis membrane at 0 h and 15 days isalso given.

FIG. 2 b is a line graph showing in vitro monomer release kinetics fromthe various supramolecular insulin assembly intermediates of bovineinsulin.

FIG. 2 c is a line graph showing in vitro monomer release kinetics fromthe various supramolecular insulin assembly intermediates of rH-insulin.

FIG. 2 d is a line graph showing in vitro release kinetics of SIA II(alternatively preamyloid) formed at pH 7.0 monitored under constant 1ml PBS solution.

FIG. 3 is a line graph showing Congo-Red binding studies with nativeinsulin, supramolecular insulin assembly II (alternatively pre-amyloidinsulin II), supramolecular insulin assembly III (alternativelypre-amyloid insulin III) and insulin amyloid (bovine and human).

FIG. 4 is a line graph showing fourier transform infrared (FTIR)spectroscopic characterization of r-human and bovine insulin.

FIG. 5 is a series of photographs showing morphologies of supramolecularinsulin assembly (alternatively pre-amyloid insulin) intermediates andinsulin fibrils studied by Atomic Force Microscopy (AFM):

(a) insulin monomer

(b) supramolecular insulin assembly I (alternatively pre-amyloid insulinI) intermediate, pH 7.0 of Bovine insulin,

(c) supramolecular insulin assembly II (alternatively pre-amyloidinsulin II), pH 7.0 of Bovine insulin,

(d) supramolecular insulin assembly intermediate III (alternativelypre-amyloid insulin III), pH 7.0 of Bovine insulin,

(e) supramolecular insulin assembly-I, pH 7.0 of rH-insulin,

(f) supramolecular insulin assembly-II, pH 7.0 of rH-insulin,

(g) supramolecular insulin assembly-III, pH 7.0 of rH-insulin,

(h) fully formed fibrils at pH 7.0, (i) shows supramolecular insulinassembly (alternatively pre-amyloid insulin) intermediate formed at 6hrs, pH 2.0 at 37° C.,

(j) amyloid fibril formed at pH 2.0,

FIG. 6 is a series of photographs showing negative staining TEMmicrographs of insulin fibrils and the intermediates during theformation of supramolecular insulin assembly

(i) Supramolecular insulin assembly I (alternatively pre-amyloid insulinI), pH 7.0,

(ii) Supramolecular insulin assembly II (alternatively pre-amyloidinsulin II), pH 7.0,

(iii) Supramolecular insulin assembly III (alternatively pre-amyloidinsulin III), pH 7.0,

(iv) Mature fibers at pH 7.0,

(v) Fiber formed at pH 2.0, 37° C.

FIGS. 7 a-b, are line graphs showing in vivo efficacy of supramolecularinsulin assembly (alternatively pre-amyloid insulin) in glucosehomeostasis.

7 a: Blood glucose level in response to various dosages ofsupramolecular insulin assembly-II (bovine) administered bothsubcutaneously and intramuscularly.

7 b: Blood glucose level in response to various dosages ofsupramolecular insulin assembly-II (r-human) administered bothsubcutaneously and intramuscularly.

FIGS. 8 a-b are line graphs showing post-prandial blood glucose levelsmonitored over a period of 135 days after administration of bovineSIA-II

8 a: Bovine insulin

8 b: rH insulin.

FIGS. 9 a-b are line graphs showing pre-prandial blood glucose levelsmonitored over a period of 160 days after administration of human SIA-II

9 a: Bovine insulin

9 b: rH insulin.

FIG. 10 is a line graph showing blood glucose level monitored afteradministration of insulin amyloid formed at pH 2.0 and 7.0.

FIG. 11 is a series of line graphs showing body weight profiles ofSIA-II treated diabetic rats, diabetic control and non-diabetic controlrats.

FIG. 12 is a line graph showing blood glucose profiles duringintraperitoneal glucose tolerance test (IPGTT)

FIG. 13 a is a line graph showing quantification of serum rH and bovineinsulin released from corresponding SIA-II using solid state ELISA inSTZ treated rats in response to supramolecular insulin assembly(alternatively pre-amyloid insulin) injected SC or IM.

FIG. 13 b is a line graph showing quantification of serum bovine insulinduring IPGTT experiment.

FIG. 13 c is a line graph showing serum rat insulin ELISA performed forIPGTT to determine the endogenous level of insulin in response toinfused glucose.

FIGS. 14 a-b are line graphs showing ¹²⁵I labeled bovine insulin SIA IItreatment of STZ diabetic rats

14 a: CPM/ml profile in serum of animals treated with 100 μg of labeledsupramolecular insulin assembly up to 25 days. The profile for 24 h isgiven in inset

14 b: Blood glucose (mg/dL) and bovine insulin (ng/ml) released in vivoprofile for 36 days. The profile for 24 h is shown in inset

FIG. 15 is a series of photographs of electrophoretic gels showingTricine-SDS-PAGE of serum from animals treated with labeled SIA II.Coommassie staning (left) and phosphor images (rest after left) of serumof 1-28 days after treatment either subcutaneously (upper panel) orintramuscularly (middle panel). The lower panel shows in vitro releasedmonomers from SIA II.

FIGS. 16 a-c are line graphs showing hyperglycemic clamp studies aftertreatment with supramolecular insulin assembly II. GIR-glucose infusionrate (mg/kg/min)

FIGS. 17 a-b are photographs showing Western blot (WB) analysis ofcultured adipocytes for insulin signaling cascade Adipocytes treatedwith (a) PBS, insulin, SIA-II, insulin released from SIA-II, (b) serumas indicated, and analysed for insulin signaling.

FIGS. 18 a-b are a series of photomicrographs showing male Wistar ratsrendered diabetic with STZ and treated subcutaneously withsupramolecular insulin assembly II (alternatively pre-amyloid insulinII) and checked for the presence of residual supramolecular insulinassembly using Congo red and the occurrence of inflammationrespectively.

18 a: Subcutaneous Congo red stained sections (i-v), Congo redBirefringence (vi-x), H & E stained sections (xi-xv), and Immunostainedsection (xvi-xx) from week 1 to week 12.

18 b: Intramuscular sections stained with Congo red (i-v), Congo redBirefringence (vi-x) H & E stained sections (xi-xv), and Immunostainedsection (xvi-xx) from week 1 to week 12.

FIGS. 19 a-b are a series of photomicrographs.

FIG. 19 a shows LPS from E coli was injected to subcutaneous and muscletissues as a positive control for infiltration of inflammatory cells.Immunostained SC (i) and IM (ii) section, and H&E stained SC (iii) andIM (iv) section.

FIG. 19 b shows insulin amyloid fibers formed at pH 2.0 injected SC weremonitored for 1, 4, 8 and 12 weeks using Congo red stained sections(i-iv) and Congo Red Birefringence (v-viii).

FIGS. 20 a-c are line graphs showing blood glucose profile of rH-insulinSIA-II treatment of rats rendered diabetic using Alloxan.

20 a: Fasting

20 b: Non-fasting Blood

20 c: Human Insulin quantification in serum using solid state ELISA asper the manufacturer's protocol.

FIGS. 21 a-e are a series of photographs showing monitoring cataractformation under the following conditions:

(a) STZ-treated Rat.

(b) Control Rat.

(c)-(d) Insulin-treated Rats.

(e) Supramolecular insulin assembly treated Rats

FIG. 22 is a series of photomicrographs showing heart, kidney and livertissue were sectioned and examined for amyloid deposition by stainingwith CR. Cellular morphology was also assessed using H&E Staining. Congored Birefringence of heart kidney and liver (i-iii), Congo red stainedsections (iv-vi), H&E staining of heart, kidney and liver (vii-ix).

FIG. 23 is a bar graph showing detection of Rat Insulin degrading enzyme(IDE) after treatment with SIA-II.

FIG. 24 is a line graph with a bar graph inset showing screening foranti-insulin antibody in Rat serum treated with SIA-II.

FIG. 25 is a bar graph showing the results of a MTT assay for cellproliferation: MCF 7 cells were assayed for their growth kinetics by theadministration of PBS, pH 7.4, human/bovine insulin (20 nM), SIA II(Human/bovine insulin), released insulin monomers from SIA II (20 nM),Insulin like Growth Factor I (IGF-1) (6 ng/ml).

FIGS. 26 a-c are line graphs showing blood glucose level in response tovarious dosages of supramolecular insulin assembly administered bothsubcutaneously and intramuscularly in mice rendered diabetic using STZ.Human SIA-II injected (a) subcutaneously, (b) intramuscularly and (c)bovine SIA-II subcutaneously at indicated dosage.

FIG. 27 is a line graph showing blood glucose level in response tosupramolecular insulin assembly administered subcutaneously in rabbitrendered diabetic using STZ.

FIGS. 28 a-b are line graphs showing blood glucose profile of Type IIdiabetic rats given various treatments as indicated in the figure, overa period of 35 days.

FIGS. 29 a-c are line graphs showing monitoring insulin counterregulatory response using Hyperinsulinemic Euglycemic/Hypoglycemic clampstudies

(a) Blood glucose level

(b) Glucagon level

(c) Epinephrine level.

FIG. 30 is a series of photographs of electrophoretic gels showing 20%coomassie stained bands showing cleavage pattern of (i) rH insulin, (ii)SIA-I, (iii) SIA-II and (iv) SIA-III with different dilutions of 2 mg/mlTrypsin and Proteinase K.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides protein therapeutics for treatment ofdiabetes and other chronic diseases/disorders. The present inventionparticularly provides supramolecular insulin assembly (SIA), wherein theSIA comprises the insoluble and aggregated oligomeric form of insulin.The present invention also provides the supramolecular exendin-4assembly (SEA), wherein the SEA comprises insoluble and aggregatedoligomeric form of exendin-4. The present invention also provides thepharmaceutical compositions comprising supramolecular insulin assemblyand/or supramolecular exendin-4 assembly.

The present invention also provides the feasibility of treating Type IIdiabetes.

The term “supramolecular insulin assembly” or “SIA” used herein refersto the insoluble and aggregated oligomeric form of insulin. The sequenceof human insulin is known (e.g., Nicol et al., Nature. 1960 Aug. 6;187:483-5) as are the sequences of insulin from other animals (e.g.,pig, mouse, dog, cat). Exendin-4 and agonists thereof are described inU.S. Pat. No. 6,506,724.

The term “supramolecular insulin assembly” or “SIA” used herein refersto the insoluble and aggregated oligomeric form of insulin, wherein thehigher order oligomeric form of SIA-II and III, prevents the proteasesto cleave them. Resistance of SIA-II and SIA-II to cleavage by Trypsinand Proteinase K further demonstrate the difference in the structuralorganization of these oligomers. rH Insulin and SIA-I are moresusceptible to cleavage by the proteases as compared to SIA-II andSIA-III, which adopt an higher order oligomeric form resistant toprotease action.

The term “supramolecular insulin assembly” or “SIA” used herein refersto supramolecular insulin assembly I, II and III, which are theinsoluble and aggregated oligomeric form of insulin.

The term “SIA I” used herein refers to “supramolecular insulin assembly”at stage I, wherein “SIA I” comprises the insoluble and aggregatedoligomeric form of insulin, wherein the oligomers consists of elongatedclusters having pearl like arrangement of insulin monomer.

The term “SIA II” used herein refers to “supramolecular insulinassembly” at stage II, wherein “SIA II” comprises the insoluble andaggregated oligomeric form of insulin, wherein the oligomers of insulinare arranged as a linear association of the above mentioned elongatedclusters, having a unique entity with a supra-oligomeric structuralorganization.

The term “SIA III” used herein refers to “supramolecular insulinassembly” at stage III, wherein “SIA III” consists of a dense, linearassociation of oligomeric form of insulin.

SIA at stage III i.e. SIA III consist of about 90% of SIA-II.

In accordance with the present invention, one embodiment provides asupramolecular insulin assembly (SIA) useful as protein therapeutics forthe treatment of metabolic disorders selected from the group consistingof type 1, type 2 diabetes mellitus and complications thereof, whereinsaid assembly comprises insoluble and aggregated oligomeric form ofinsulin.

In another embodiment of the present invention, there is provided asupramolecular insulin assembly (SIA) useful as protein therapeutics forthe treatment of metabolic disorders selected from the group consistingof type 1, type 2 diabetes mellitus and complications thereof, whereinthe assembly comprises insoluble and aggregated oligomeric form ofinsulin as SIA I, SIA II, SIA III or combination thereof, wherein SIA Iconsists of elongated clusters having pearl like arrangement of insulinmonomer, SIA II consists of linear association of elongated clustershaving pearl like arrangement of insulin monomer and SIA III consists ofabout 90% SIA-II, that is a dense, linear association of insulinoligomers.

In another embodiment of the present invention, there is provided asupramolecular insulin assembly (SIA) useful as protein therapeutics forthe treatment of metabolic disorders selected from the group consistingof type 1, type 2 diabetes mellitus and complications thereof, whereinthe assembly comprises insoluble and aggregated oligomeric form ofinsulin as SIA I, wherein SIA I consists of elongated clusters havingpearl like arrangement of insulin monomer.

In yet another embodiment of the present invention, there is provided asupramolecular insulin assembly (SIA) useful as protein therapeutics forthe treatment of metabolic disorders selected from the group consistingof type 1, type 2 diabetes mellitus and complications thereof, whereinthe assembly comprises insoluble and aggregated oligomeric form ofinsulin as SIA II, wherein SIA II consists of linear association ofelongated clusters having pearl like arrangement of insulin monomer.

In still yet another embodiment of the present invention, there isprovided a supramolecular insulin assembly (SIA) useful as proteintherapeutics for the treatment of metabolic disorders selected from thegroup consisting of type 1, type 2 diabetes mellitus and complicationsthereof, wherein the assembly comprises insoluble and aggregatedoligomeric form of insulin as SIA III, wherein SIA III consists dense,linear association of insulin oligomers.

One embodiment provides the supramolecular insulin assembly (SIA),wherein the insulin is recombinant human insulin, bovine or pig insulin,or mutants/analogs of insulin.

One embodiment of the present invention provides the supramolecularinsulin assembly (SIA), wherein the assembly comprises insoluble andaggregated oligomeric form of insulin, wherein the assembly releaseinsulin at a rate ranging from about 0.2 to 0.6 IU per hour in vitro.

One embodiment of the present invention provides the supramolecularinsulin assembly (SIA), wherein the assembly comprises insoluble andaggregated oligomeric form of insulin as SIA I, SIA II, SIA III orcombination thereof, wherein SIA I consists of elongated clusters havingpearl like arrangement of insulin monomer, SIA II consists of linearassociation of elongated clusters having pearl like arrangement ofinsulin monomer and SIA III consists of about 90% SIA-II, that is adense, linear association of insulin oligomers,

wherein the assembly release insulin at a rate ranging from about 0.2 to0.6 IU per hour in vitro.

One embodiment of the present invention provides the supramolecularinsulin assembly (SIA), wherein the assembly comprises insoluble andaggregated oligomeric form of insulin, wherein the assembly releasesinsulin at a rate ranging from 0.1 to 5.4 ng/ml, wherein rate of releaseof insulin is in the range of for about 7 to 180 days, in vivo.

One embodiment of the present invention provides the supramolecularinsulin assembly (SIA), wherein the assembly comprises insoluble andaggregated oligomeric form of insulin as SIA I, SIA II, SIA III orcombination thereof, wherein SIA I consists of elongated clusters havingpearl like arrangement of insulin monomer, SIA II consists of linearassociation of elongated clusters having pearl like arrangement ofinsulin monomer and SIA III consists of about 90% SIA-II, that is adense, linear association of insulin oligomers, wherein the assemblyreleases insulin at a rate ranging from 0.1 to 5.4 ng/ml, wherein rateof release of insulin is in the range of for about 7 to 180 days, invivo.

One embodiment of the present invention provides the supramolecularinsulin assembly (SIA), wherein the assembly comprises insoluble andaggregated oligomeric form of insulin, wherein the assembly releaseinsulin at a rate ranging from about 0.2 to 0.6 IU per hour in vitro,wherein rate of release of insulin is in the range of 4-5.4 ng/ml for atleast 7-10 days.

One embodiment of the present invention provides the supramolecularinsulin assembly (SIA), wherein the assembly comprises insoluble andaggregated oligomeric form of insulin as SIA I, SIA II, SIA III orcombination thereof, wherein SIA I consists of elongated clusters havingpearl like arrangement of insulin monomer, SIA II consists of linearassociation of elongated clusters having pearl like arrangement ofinsulin monomer and SIA III consists of about 90% SIA-II, that is adense, linear association of insulin oligomers, wherein the assemblyrelease insulin at a rate ranging from about 0.2 to 0.6 IU per hour invitro, wherein rate of release of insulin is in the range of 4-5.4 ng/mlfor at least 7-10 days.

In yet another embodiment of the present invention, there is provided asupramolecular insulin assembly (SIA) useful as protein therapeutics forthe treatment of metabolic disorders selected from the group consistingof type 1, type 2 diabetes mellitus and complications thereof, whereinthe assembly comprises insoluble and aggregated oligomeric form ofinsulin as SIA II, wherein SIA II consists of linear association ofelongated clusters having pearl like arrangement of insulin monomer,wherein rate of release of insulin is in the range of 0.5-1.8 ng/ml forat least 160 days.

In still yet another embodiment of the present invention, there isprovided a supramolecular insulin assembly (SIA) useful as proteintherapeutics for the treatment of metabolic disorders selected from thegroup consisting of type 1, type 2 diabetes mellitus and complicationsthereof, wherein the assembly comprises insoluble and aggregatedoligomeric form of insulin as SIA III, wherein SIA III consists dense,linear association of insulin oligomers, wherein rate of release ofinsulin is in the range of 0.1-0.7 ng/ml for at least 180 days.

In yet another embodiment of the present invention, there is provided asupramolecular insulin assembly (SIA) useful as protein therapeutics forthe treatment of metabolic disorders selected from the group consistingof type 1, type 2 diabetes mellitus and complications thereof, whereinthe assembly comprises insoluble and aggregated oligomeric form ofinsulin as SIA II, wherein SIA II consists of linear association ofelongated clusters having pearl like arrangement of insulin monomer,wherein the assembly upon administration to diabetic subjects maintainsnear-normoglycemic level (120±30 mg/dl) for at least 160 days in asubject in need thereof.

The supramolecular insulin assembly (SIA) as disclosed in the presentinvention, wherein a single dose of the assembly upon administrationmaintains near-normoglycemic level (120±30 mg/dl) for at least 7 to 180days in a subject in need thereof, wherein concentration of the assemblyin the dose is in the range of 25 to 750 μg.

The supramolecular insulin assembly (SIA) as disclosed in the presentinvention, wherein a single dose of the assembly upon administrationmaintains near-normoglycemic level (120±30 mg/dl) for at least 160 daysin a subject in need thereof, wherein concentration of the assembly inthe dose is in the range of 150 to 250 μg.

One embodiment of the present invention provides a supramolecularexendin-4 assembly (SEA) useful as protein therapeutics for thetreatment of diabetes, wherein the assembly comprises insoluble andaggregated oligomeric form of exendin-4.

Another embodiment of the present invention provides the supramolecularinsulin assembly, wherein the assembly is a non cytotoxic, nonimmunogenic, non-apoptotic and non-mitogenic prodrug.

Another embodiment of the present invention provides the supramolecularinsulin assembly, wherein the assembly is a non cytotoxic, nonimmunogenic, non-apoptotic and non-mitogenic prodrug.

One embodiment of the present invention provides a pharmaceuticalcomposition for the treatment of metabolic disorders selected from thegroup consisting of type 1, type 2 diabetes mellitus and complicationsthereof, the composition comprising therapeutically effective amount ofthe supramolecular insulin assembly (SIA) as disclosed in the presentinvention.

Another embodiment of the present invention provides a pharmaceuticalcomposition for the treatment of metabolic disorders selected from thegroup consisting of type 1, type 2 diabetes mellitus and complicationsthereof, the composition comprising therapeutically effective amount ofthe supramolecular insulin assembly-II (SIA-II).

Yet another embodiment of the present invention provides apharmaceutical composition for the treatment of metabolic disordersselected from the group consisting of type 1, type 2 diabetes mellitusand complications thereof, the composition comprising therapeuticallyeffective amount of the supramolecular insulin assembly (SIA) andsupramolecular exendin-4 assembly (SEA).

Still yet another embodiment of the present invention provides apharmaceutical composition for the treatment of metabolic disordersselected from the group consisting of type 1, type 2 diabetes mellitusand complications thereof, the composition comprising therapeuticallyeffective amount of the supramolecular insulin assembly (SIA) andsupramolecular exendin-4 assembly (SEA).

Further embodiment of the present invention provides a pharmaceuticalcomposition for the treatment of metabolic disorders selected from thegroup consisting of type 1, type 2 diabetes mellitus and complicationsthereof, the composition comprising therapeutically effective amount ofsupramolecular exendin-4 assembly (SEA).

The pharmaceutical composition(s) disclosed in the present inventionoptionally comprises pharmaceutically acceptable carriers, additives ordiluents.

The pharmaceutical composition(s) disclosed in the present invention isadministered intramuscularly, intradermally or subcutaneously.

The pharmaceutical composition(s) disclosed in the present invention isadministered through a device capable of releasing the said composition,wherein said device is selected from a group consisting of pumps,catheters, patches and implants.

In one embodiment, there is provided a process of preparation ofsupramolecular insulin assembly (SIA), the process comprising dissolvinginsulin at a temperature of about 25 to 60° C. in a solution having pHin the range of about 1.5 to 7.8; and incubating the above for a periodof about 6 to 48 hours with constant shaking to obtain SupramolecularInsulin Assembly (SIA), wherein SIA comprises insoluble and aggregatedoligomeric form of insulin

The process of preparation of supramolecular insulin assembly (SIA)disclosed in the present invention, wherein the process furthercomprises washing the SIA with PBS; and re-suspending the washed SIA inPBS.

The process of preparation of supramolecular insulin assembly (SIA)disclosed in the present invention, wherein incubation period is 10hours.

In another embodiment of the present invention, there is provided aprocess of preparation of supramolecular exendin-4 assembly (SEA), theprocess comprising dissolving exendin-4 at a temperature of about 25 to60° C. in a solution having pH in the range of about 2.0 to 7.6; andincubating the above for a period of 6 to 192 hours with constantshaking to obtain Supramolecular Exendin-4 Assembly (SEA), wherein SEAcomprises insoluble and aggregated oligomeric form of Exendin-4

The process of preparation of supramolecular exendin-4 assemblydisclosed in the present invention further comprises washing the SEAwith PBS; and re-suspending the washed SEA in PBS.

The process of preparation of supramolecular exendin-4 assembly (SEA)disclosed in the present invention, wherein the incubation period is 148hours.

The process preparation of the supramolecular insulin assembly (SIA)disclosed in the present invention, wherein the solution is selectedfrom a group consisting of hydrochloric or acetic acid in water havingpH in the range of about 1.5 to 2.5; sodium acetate buffer having pH inthe range of about 3.5 to 5.5; phosphate buffer (PBS) having pH 6-7.5,and citrate buffer having pH in the range of about 4 to 6.

The process preparation of supramolecular exendin-4 assembly (SEA)disclosed in the present invention, wherein the solution is selectedfrom a group consisting of hydrochloric or acetic acid in water havingpH in the range of about 1.5 to 2.5; sodium acetate buffer having pH inthe range of about 3.5 to 5.5; phosphate buffer (PBS) having pH 6-7.5,and citrate buffer having pH in the range of about 4 to 6.

The process preparation of the supramolecular insulin assembly (SIA)disclosed in the present invention, wherein the temperature is 37° C.

The process preparation of supramolecular exendin-4 assembly (SEA)disclosed in the present invention, wherein the temperature is 37° C.

The process preparation of the supramolecular insulin assembly (SIA)disclosed in the present invention, wherein pH of said solution is 7.2.

The process preparation of supramolecular exendin-4 assembly (SEA)disclosed in the present invention, wherein pH of said solution is 7.2.

The process preparation of the supramolecular insulin assembly (SIA)disclosed in the present invention, wherein said period is 6-192 hours.

The process preparation of the supramolecular insulin assembly (SIA)disclosed in the present invention, wherein said period is 6-192 hours.

One embodiment of the present invention relates to a method for treatingmetabolic disorders selected from the group consisting of type 1, type 2diabetes mellitus and complications thereof, wherein the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of the pharmaceutical composition comprising thesupramolecular insulin assembly, which is effective for the alleviationof the disorder.

Yet another embodiment of the present invention provides a method fortreating metabolic disorders selected from the group consisting of type1, type 2 diabetes mellitus and complications thereof, wherein themethod comprising administering to a subject in need thereof atherapeutically effective amount of the pharmaceutical compositioncomprising the supramolecular insulin assembly-II (SIA-II), which iseffective for the alleviation of the disorder.

Still yet another embodiment of the present invention provides a methodfor treating metabolic disorders selected from the group consisting oftype 1, type 2 diabetes mellitus and complications thereof, wherein themethod comprising administering to a subject in need thereof atherapeutically effective amount of the pharmaceutical compositioncomprising the supramolecular exendin-4, which is effective for thealleviation of the disorder.

Still yet another embodiment of the present invention provides a methodfor treating metabolic disorders selected from the group consisting oftype 1, type 2 diabetes mellitus and complications thereof, wherein themethod comprising administering to a subject in need thereof atherapeutically effective amount of the pharmaceutical compositioncomprising the supramolecular insulin assembly and supramolecularexendin-4 assembly, which is effective for the alleviation of thedisorder.

In further embodiment of the present invention, there is provided use ofsupramolecular insulin assembly for the treatment of metabolic disordersselected from the group consisting of type 1, type 2 diabetes mellitusand complications thereof.

In yet another embodiment of the present invention, there is provideduse of supramolecular exendin-4 assembly for the treatment of metabolicdisorders selected from the group consisting of type 1, type 2 diabetesmellitus and complications thereof.

In still yet another embodiment of the present invention, there isprovided use of supramolecular insulin assembly in combination withsupramolecular exendin-4 assembly for the treatment of metabolicdisorders selected from the group consisting of type 1, type 2 diabetesmellitus and complications thereof.

In additional embodiment, there is provided the supramolecular insulinassembly (SIA), wherein the assembly shows sharp peak at 1647-1645 cm⁻¹in Fourier Transform infrared spectroscopy (FTIR).

In one embodiment, the present invention provides the supramolecularinsulin assembly (SIA), wherein the insulin is recombinant Humaninsulin.

In another embodiment, the present invention provides the supramolecularinsulin assembly (SIA), wherein the insulin is human, bovine or piginsulin.

In yet another embodiment of the present invention there is provided thesupramolecular insulin assembly (SIA), wherein the assembly uponadministration releases insulin monomers.

Further the present invention provides the supramolecular proteinassembly, wherein the peptide in assembly is coupled with a smallmolecule drug.

One embodiment of the present invention provides the supramolecularprotein assembly disclosed in the present invention acts as a prodrug,

The pharmaceutical composition disclosed in the present inventioncomprises pharmaceutically acceptable carriers, additives or diluents.

The pharmaceutical composition disclosed in the present invention isadministered intramuscularly or subcutaneously.

The pharmaceutical composition disclosed in the present invention isadministered through a device capable of releasing the composition,wherein the device is selected from a group consisting of pumps,catheters and implants.

The pharmaceutical composition disclosed in the present invention,wherein single dose of the composition upon administration releases saidprotein for prolonged period.

The process of preparation of supramolecular insulin assembly disclosedin the present invention comprises incubation of the insulin in thesolution for 10 hours.

The process of preparation of supramolecular protein assembly disclosedin the present invention comprises incubation of the protein in thesolution for 6-192 hours.

The method for treatment using the pharmaceutical composition asdisclosed in the present invention, wherein the composition isadministered intramuscularly, intra-peritonealy or subcutaneously.

In still another embodiment, the present invention provides acomposition comprising of the supramolecular insulin assembly (SIA)which is stable, protease resistant and has longer shelf life.

In still another embodiment, the present invention provides acomposition comprising of the exendin-4 assembly (SEA) which is stable,protease resistant and has longer shelf life.

In still another embodiment, the present invention provides acomposition comprising of the supramolecular insulin assembly (SIA)which is stable, protease resistance and has longer shelf life rangingfrom about 10 days to 150 days or more.

In still another embodiment, the present invention provides acomposition comprising of the supramolecular exendin-4 assembly (SEA)which is stable, protease resistance and has longer shelf life rangingfrom about 10 days to 150 days or more.

As will be appreciated by those in the art a variety of the solutionssuch as known buffers can be used for re-suspension and washing of thesupramolecular protein assembly disclosed in the present invention.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent to treat, ameliorate, or prevent a desireddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. The precise effective amount for a subject willdepend upon the subject's size and health, the nature and extent of thecondition, and the therapeutics or combination of therapeutics selectedfor administration. The effective amount for a given situation isdetermined by routine experimentation and is within the judgment of theclinician.

For purposes of the present invention, an effective dose of SIA willgenerally be from about 0.1 mg/kg to about 1.0 mg/kg, or about 0.2 mg/kgto about 2.0 mg/kg or about 0.5 mg/kg to about 3.0 mg/kg of thecompositions of the present invention in the subject to which it isadministered.

A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which can be administered without undue toxicity.Suitable carriers can be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Pharmaceutically acceptable carriers in therapeuticcompositions can include liquids such as water, saline, glycerol andethanol. Auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, can also be present in suchvehicles. Typically, the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection can also be prepared. Liposomes and neosomes are includedwithin the definition of a pharmaceutically acceptable carrier.Pharmaceutically acceptable salts can also be present in thepharmaceutical composition, e.g., mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

The compositions disclosed in the present invention comprisesupramolecular protein assemblies of the relevant/applicable therapeuticproteins and are applicable for treatment of a number of chronicdiseases and acute symptoms.

The compositions disclosed in the present invention comprises oligomersof therapeutic proteins particularly the supramolecular assembly of aprotein for sustained release of the protein.

Some widely-used biopharmaceuticals such as insulin, glucagon, andcalcitonin can be induced to form amyloids. Compared to solubleprecursor proteins, amorphous aggregates formed as a prelude to amyloidformation, gain new properties such as enhanced stability, proteaseresistance, self-propagation, longer shelf life, highly organizedstructure and can serve as a concentrated compact source of puremolecules.

The supramolecular insulin assemblies disclosed in the present inventionexist with in a defined structure having both α-helical and β-sheetcomponents. The adoption of the above mentioned unique structure resultsin a change in its solubility and its structure from the native insulinmolecules in solution. Significantly, release of insulin monomers fromthis supramolecular structure are biologically active and thus resemblethe native insulin structure. Novel dissolution properties are conferredupon this supramolecular structure formed, which acts as a prodrug, asby itself viz. In its prodrug form it was found that it has no action oreffect on the signaling events or glucose homeostasis of the insulinsensitive fat cells. It is transformed into a drug by the release ofinsulin monomers from the depot or site of injection in vivo. Both invitro and in vivo results demonstrate the uniqueness of the formulationto release bioactive insulin molecules showing an effect on the glucosehomeostasis and other clinical parameters usually assessed for type Iand II diabetes.

The present invention provides a composition comprising supramolecularinsulin assembly useful in the treatment of diabetes mellitus.Supramolecular insulin is a hybrid of amorphous and nascent fibrillaroligomers of insulin and serves as a sustained release formulation forthe release of bioactive insulin. The glucose regulatory hormone insulinis a 51 residue polypeptide that exists in equilibrium as a mixture ofdifferent oligomeric states, including hexamers, dimers and monomers,depending on the environment. In pancreatic secretary vesicles, insulinis stored as a hexamer at physiological pH, whereas it interacts withits receptor as a monomer. Under denaturing conditions, such as low pHor in the presence of strong denaturants, insulin aggregates to formamyloid fibrils (Paul Bevan Insulin signalling. J. Cell Sci., 114,1429-1430 (2001)).

The supramolecular oligomers of insulin disclosed in the presentinvention is prepared at a pH ranging from 6.8 to 7.8 preferably 7.2.

The present invention provides a composition comprising supramolecularinsulin assembly capable of sustained release of insulin monomers. Thecomposition comprising a given supramolecular insulin assembly (viz SIAI, II, III or a combination thereof) is useful for achieving betterglycemic control.

The present invention provides insulin oligomers, supramolecular insulinassembly II that is useful in achieving tighter glycemic control byachieving a sustained release of insulin. Supramolecular insulinassembly II when administered subcutaneously or intramuscularlymaintains a basal level of insulin in STZ induced diabetic rat for aprolonged period ranging from about 10 days to 180 days or more, whilesimultaneously keeping a tight glycemic control, thus affording a longlasting treatment against diabetes mellitus.

According to the present invention, one embodiment provides acomposition comprising supramolecular insulin assembly II that causes asustained release of insulin monomers ranging between 0.5-1.5 ng/ml andlasts about at least 180 days when administered intramuscularly orsubcutaneously.

Another embodiment of the present invention provides that thecomposition comprising supramolecular insulin assembly II wherein theamount of insulin monomer released from the supramolecular insulinassembly II is in the range of 0.5-1.5 ng/ml in serum.

In an embodiment of the present invention, the in-vivo effect of thecomposition comprising supramolecular oligomers of insulin oncontrolling blood glycemic levels have been verified using STZ induceddiabetic rats.

Another embodiment of the present invention provides the dosage of thecomposition comprising supramolecular insulin assembly wherein thedosage ranging from about 50 μg to about 400 μg of insulin was monitoredfor the experimental time period.

Another embodiment of the present invention provides the dosage of thecomposition comprising supramolecular insulin assembly wherein thedosage is 200 μg of insulin.

Another embodiment of the present invention provides the dosage of thecomposition comprising supramolecular insulin assembly wherein thedosage is 100 μg of insulin.

In still another embodiment of the present invention provides acomposition comprising the supramolecular insulin assembly, wherein thecomposition does not elicit insulin degrading enzyme activity (IDE) inand around the site of injection for a time period ranging from about 10days to 180 days or more.

In yet another embodiment of the present invention, there is absence ofanti-insulin antibodies in the serum of the subject for a time period ofranging from about 10 days to 150 days or more.

In still another embodiment, the present invention provides acomposition comprising of the supramolecular insulin assembly which isstable, protease resistance and has longer shelf life ranging from about10 days to 150 days or more.

In yet another embodiment of the present invention there is provided acomposition comprising the supramolecular insulin assembly which iscapable of releasing insulin monomers in a controlled manner for a longperiod of time without any burst of rapid release. The kinetics oftransformation can be controlled both in vivo and in vitro.

Further, a composition comprising the supramolecular insulin assemblydisclosed in the present invention can be used as a single dose havinglong lasting effect that frees the patients from the need to administermultiple doses of insulin every day.

The composition comprising the supramolecular insulin assembly disclosedin the present invention does not exhibit an abrupt large release ofinsulin thwarting hypoglycemic stage in diabetic subjects.

The composition comprising the supramolecular insulin assembly disclosedin the present invention is capable of releasing insulin monomers at aconstant rate both in vitro and in vivo.

In still another embodiment of the present invention, the higheroligomeric stage of the supramolecular insulin assembly achieves atightly regulated glycemic control without fasting hypoglycemia indiabetic subject.

In still another embodiment of the present invention, the higheroligomeric stage of the supramolecular insulin assembly achieves atightly regulated glycemic control without fasting hypoglycemia in Type1 diabetic subject.

In still another embodiment of the present invention, the higheroligomeric stage of the supramolecular insulin assembly achieves atightly regulated glycemic control without fasting hypoglycemia in Type2 diabetic subject.

In yet another embodiment of the present invention, there is no suddenincrease of body weight of subjects when treated with the supramolecularinsulin assembly or composition comprising supramolecular insulinassembly of the present invention.

In still another embodiment of the present invention, no lag phase isfound in the release of bioactive insulin monomers from thesupramolecular insulin assembly II in the Intraperitoneal GlucoseTolerance Test (IPGTT) blood glucose profile of the subject, similar tothe administration of free insulin.

In yet another embodiment of the present invention, zero order kineticsor sustained release is observed for in vivo release of insulin monomersfrom the supramolecular insulin assembly II.

In still another embodiment of the present invention, insulin releasedfrom the supramolecular insulin assembly II is equivalent in biologicalfunction to soluble insulin.

Further, in another embodiment of the present invention, toxicity of thesupramolecular insulin assembly is ruled out by performing biologicalassays for Serum glutamate oxalo-acetate transaminase (SGOT), serumglutamate pyruvate transaminase (SGPT), total Bilirubin, Bilirubin,Alkaline Phosphatase, Serum total proteins, Serum Albumin, SerumGlobulin, Serum A/G ratio, Kidney function test (KFT), CataractFormation, Adipose Tissue weight, Body Weight and appearance.

In yet another embodiment, the rate of glucose infusion remains the samein treated animals. This concludes that the monomers released from thedepot at the site of injection are biologically active and stimulate themuscles and the liver to take up glucose.

In still another embodiment, MTT assay was performed on MCF 7 cells tovalidate the unchanged structural and binding dynamics of the insulinmonomers, released from SIA II.

Further, in another embodiment of the present invention, male Wistarrats were rendered diabetic using another chemical compound Alloxan.These diabetic rats were further treated with SIA II and showed similarresult to that of STZ induced diabetic rat. A tight glycemic control wasobserved.

In still another embodiment of the present invention, C57BL/6 mice weremade diabetic using streptozotocin, thereafter treated with SIA II, alsoshowed normo-glycemic levels.

Another embodiment of the present invention is that the supramolecularinsulin assembly is a stable depot useful for the controlled release ofactive peptide drugs from the supramolecular insulin assembly termini.

According to one embodiment of the present invention the supramolecularinsulin assembly II is capable of affording a long lasting treatmentagainst diabetes.

In another embodiment, the insulin used for preparation ofsupramolecular insulin assembly is preferably recombinant human insulin,bovine and pig insulin.

In still another embodiment, the composition comprising thesupramolecular insulin assembly I and II (SIA I and II) or a combinationthereof, disclosed in the present invention is capable of lowering bloodglucose levels in animal subjects treated with streptozotocin to inducetype II diabetes.

In still another embodiment, SIA I, which releases insulin monomers at afaster rate was administered to rats suffering from Diabetes MellitusType II (DM II).

According to one embodiment of the present invention, SIA II was alsoadministered to DM II rats for the treatment.

In yet another embodiment, diabetic rats treated with both SIA I and IIshowed near normoglycemic levels bordering on the higher side, in bothpre-prandial and post-prandial (180±15 mg/dl) states.

In yet another embodiment of the present invention, the dosage of SIA IIinjected was 150 μg in two places, both subcutaneously andintramuscularly.

Another embodiment of the present invention demonstrates the ability ofSIA I and II to maintain near normoglycemic levels for up to 30 days.

In still another embodiment, administration of Exendin 4 along withinsulin therapy was able to maintain near normoglycemic levels (135±10mg/dl) for up to 45 days, and was a better formulation for treating DMII.

Further in another embodiment, SIA III, which releases insulin monomersbut at a very slow rate (0.05-0.3 ng/ml) which can be useful in thetreatment of borderline diabetic patients, viz prediabetic or subjectswith poor prognosis in glucose tolerance tests, who require very littleamount of insulin as a therapy.

In yet another embodiment of the present invention, the level of humaninsulin detected in the serum of diabetic rats is about 0.7-0.85 ng/ml.

In still another embodiment of the present invention, various serumparameters were estimated, such as triglycerides (TAG), free fatty acids(FFA), to demonstrate the effectiveness of the treatment.

In yet another embodiment, the level of TAG in the serum was in therange of 0.45-0.6 mmol/l for the Exendin 4 treated rats, which is almostsame to the control.

Further in another embodiment, the FFA levels in the serum was estimatedto be about 0.85-0.9 mmol/l for the Exendin 4a treated rats, which isalmost same to the control.

In still another embodiment of the present invention, the increase inthe body weight of the treated animals was almost same as that ofcontrol rats.

In yet another embodiment, the current methodology can be extended tothose chronic and inflammatory diseases where a sustained and continuoustherapy is required using peptides, proteins or small molecules.

Example 1 and 2 of the present invention provides a process forpreparation of supramolecular insulin assembly II. The FIG. 1 providesdetails of the insulin (both human and bovine) fibril formationmonitored by using Thioflavin T (Th-T) fluorescence (Le Vine, H.Quantification of β-Sheet Amyloid Fibril Structures with Thioflavin T.Methods Enzymol 309, 274-284 (1999)). Fibril formation by bovine insulinwas noted by acquisition of Th-T fluorescence, when the insulin wasagitated at 180 rpm at 37° C., both at pH 7.0 (50 mM PBS) and pH 2.0(hydrochloric acid in water) (FIG. 1). The increase in Th-T fluorescenceoccurred due to its binding to insulin fibrils reaching a maximum valueat 48 hrs for insulin incubated at pH 7.0, while at pH 2.0 the fibrilformation is rapid and therefore, Th-T fluorescence attained a maximumvalue at 20 hr. FIG. 1 compares the fibril formation by both bovine andhuman insulin.

Example 3 of the present invention provides kinetics of the release ofinsulin monmers from supramolecular insulin assembly-II. Thesupramolecular insulin assembly form of insulin acts as a reservoir forthe sustained release of insulin monomer for a long time (FIG. 2 a). Therelease of insulin monomers from its amyloid and various supramolecularassemblies of insulin were noted and is depicted by FIG. 2 b & c. Thefully formed amyloid fibers release negligible insulin irrespective ofpH. At pH 2.0, supramolecular insulin assembly intermediates,particularly, supramolecular insulin assembly II, release insulin at avery slow rate suggesting that these oligomers are sturdy and tightlyassociated. However, the pH 7.0 intermediate (termed supramolecularinsulin assembly II), exhibiting a turbidity of 0.9-1.3 at 600 nm,release insulin at an appreciable rate. A linear increase in the releaseof insulin monomers at 280 nm absorbance over a period of 15±5 days isobserved. When insulin release from SIA was observed under constantvolume of 1 ml, a bell shaped curve was observed (FIG. 2 d). Thisdemonstrates the reversibility of the oligomer formation procedure andthe existence of equilibrium between free insulin and SIA. The tyrosinefluorescence of the released sample corroborated these observations asis provided in FIG. 2 a. The insulin amyloid intermediate,supramolecular insulin assembly II (alternatively called insulinpre-amyloid II) meets the requirement of a sustained release of 2-4 μM(0.4-0.6 IU) of insulin monomers per hour in vitro, thereby achieving aconstant yet slow release in vivo for maintaining basal insulin levelsin the body.

Example 4 provides characterization of supramolecular insulin assemblyusing Congo-Red (CR) binding (Klunk, W. E., Jacob, R. F. & Mason, R. P.Quantifying amyloid by Congo red spectral shift assay. Methods Enzymol309, 285-305 (1999)). Like Th-T, CR also binds specifically to theβ-sheet rich structures of amyloid and has been used routinely for theirdetection. CR binding to samples incubated with 50 μM CR in PBS for 1 hat 37° C. was monitored by the red shift in its absorption maximum byscanning 400-600 nm regions. FIG. 3 provides that the supramolecularinsulin assembly (rH and bovine) exhibits weak binding to CR, whereasfully grown fibers at pH 2.0 and 7.0 showed significant binding.

Example 5 provides tyrosine fluorescence study. Example 6 providesFourier Transform infrared spectroscopy study. Supramolecular insulinassembly I, II and III were also characterized using ATR-FTIR. Distinctspectra corresponding to each stage was observed (FIG. 4). A shift ofthe IR band towards lower frequencies is observed. Supramolecularinsulin assembly II has a sharp peak at 1647-1645 cm⁻¹, while the fullyformed insulin fibril (amyloid) has a peak at 1630-1628 cm⁻¹ for bovineand rH insulin respectively. The FTIR spectra of Supramolecular insulinassembly II is in good agreement with the CR data showing that theprotein is still largely helical, albeit with an increase in the contentof random coil structure.

Morphology of fibers formed at pH 2.0 and 7.0 were assessed by AtomicForce Microscopy (AFM) and Transmission Electron Microscopy (TEM) asprovided in Example 7 and 8 respectively. Native insulin molecules (bothbovine and rH) at pH 7.0 and pH 2.0 show random distribution with aheight of 1.3±0.21 nm, which correlates with the dimension of insulinmonomers (1.11 nm) and dimers (1.49 nm) (FIG. 5( i)). Intermediates ofthe fibrilization process at pH 7.0 are shown in FIG. 5( ii-iv) forbovine insulin and FIG. 5( v-vi) for rH insulin. SIA-I representelongated clusters having a pearl-like arrangement. In between, thereare some elongated linear particles with 12±2 nm heights, suggestingfurther association into higher oligomeric states. The SIA-IIintermediate is seen as a linear association of the above mentionedelongated clusters in case of both bovine and rH insulin, having aunique entity with a supra-oligomeric structural organization. SIA-III(fibril stage succeeding SIA-II) showed an increase in density of higheroligomeric structures. Fully grown fibers at pH 7.0 represent thetypical cross β structure of insulin amyloid (FIG. 5( vii)).Intermediates of pH 2.0 fibrilization process at 6-7 h reveal a lateralassociation of two fibrils of height 7.2-8.3 nm, and after 20 h, largetwisted fibers of 10-12 nm width were observed (FIG. 5 ix and 5 x).

Example 8 provides Transmission Electron Microscopy (TEM) to assess thepresence of the possible assemblies of insulin. TEM micrographs showsthe presence of some amorphous and higher oligomeric structures in thesupramolecular insulin assembly II stage (FIG. 6 i). The stages aftersupramolecular insulin assembly II (FIG. 6 ii) have more fibrillarstructure as shown in (FIG. 6 iii & iv)). In contrast, there is anabundance of fibrillar forms when insulin was incubated at pH 2.0 (FIG.6 v).

Example 9 provides details of the animal models used for studying theeffectiveness of insulin amyloid and supramolecular insulin assemblyforms. The authors have used four different diabetes models for testingthe hypothesis and the therapeutic potential of SIA, in the treatment ofdiabetes, namely, (a) STZ treated rats, (b) Alloxan treated rats and (c)STZ treated mice (d) STZ rabbit

To standardize the dosage, 50 μg, 100 μg, 200 μg and 400 μg ofsupramolecular insulin assembly-II was injected subcutaneously andalternatively intramuscularly in diabetic rats. As summarized in FIG. 7a&b, a single dose of 50 and 100 μg of supramolecular insulin assemblymaintained near-normoglycemic levels up to only 10 and 30 daysrespectively, compared to 135 and 160 days when 200 μg supramolecularinsulin assembly, both bovine and human respectively was used. In caseof 400 μg dosage, sudden non-fasting normoglycemia and fastinghypoglycemia was observed irrespective of the route used, suggestingrelease of an initial bolus of a basal level of insulin monomer from theSIA depot of bovine insulin. The usage of 200 μg of supramolecularinsulin assembly as a therapeutic dosage was chosen for the detailedlong term prospective studies.

Example 10 provides the treatment of the diabetic animals withsupramolecular insulin assembly form of insulin. The STZ-induceddiabetic rats assigned to multiple groups were treated with insulin (4IU/kg, IP), supramolecular insulin assembly (200 μg both SC and IM),respectively, to study the in vivo effect of the supramolecular insulinassembly therapy on controlling blood glycemic levels. In addition, onegroup of PBS treated diabetic rats and another group of normal ratsserved as diabetic and non-diabetic control. The pre-prandial andpost-prandial blood glucose levels of the rats were monitored for aperiod till the physiological effect of insulin was observed. As shownin FIG. 8 a&b, treatment with 200 μg of supramolecular insulin assemblymaintained the basal level of insulin by the sustained and slow releaseof insulin monomers from supramolecular insulin assembly depot inSTZ-induced diabetic rats, leading to a significant reduction in thenon-fasting blood glucose levels without fasting hypoglycemia (FIG. 9a&b). No significant difference was observed whether supramolecularinsulin assembly was injected via subcutaneous or intramuscular route.In the case of free insulin (viz. in its normal counterpart;non-supramolecularly organized form) treatment (single dose of 4 IU/kg),daily blood glucose was reduced to a non-fasting value of 350-450 mg/dland fasting 300-400 mg/dl only. Diabetic rats were given insulinintraperitoneally everyday maintained hyperglycemic blood glucose levels(300±100 mg/dl). In contrast to free insulin treatment, where daily dosewas required to keep blood glucose from plummeting to very high levels,single injection of supramolecular insulin assembly was sufficient forachieving near-normoglycemia levels (150±60 mg/dl) in diabetic rat for 3months without fasting hypoglycemia (90±50 mg/dl). Similar results wereobtained with supramolecular insulin assembly formed from human insulin,as depicted in FIGS. 8 b&9 b. Administration of insulin amyloid, formedat pH 7.0 and 2.0, had no beneficial effect on the glycemic status ofdiabetic rats, as evident from FIG. 10. Again the body weight which isan indicator of normal health was monitored along with BGL in all casesover the entire period of treatment. As shown in FIG. 11, there was aninitial loss of body weight immediately after STZ treatment, butprogressive weight gains were achieved in diabetic rat after treatmentwith supramolecular insulin assembly II and the curve of supramolecularinsulin assembly treated animals paralleled that of non-diabeticcontrol. Thus, the surprising efficacy of supramolecular insulinassembly II for use in long term treatment of diabetes mellitus isconsidered an improvement over the conventional methods.

Example 11 provides intraperitoneal glucose tolerance test to assess theefficacy of supramolecular insulin assembly to release insulin in acontinuous manner. The in vivo release of insulin was monitored. Thebiological activity of the released monomer was estimated by bloodglucose disposal from the blood. The details of the independentexperiments performed are shown in FIG. 12. The glucose levels weredetermined before (TO) and 30, 90, 150, 270 and 330 min after glucoseadministration to fasting normal and STZ treated rats, upon treatmentwith PBS, supramolecular insulin assembly II and insulin. As shown inFIG. 12, insulin injection, supramolecular insulin assembly II treatmentor in vitro released monomers alone significantly improved the glucosetolerance test in treated animals, and their elevated blood glucoselevels after the glucose infusion returned to the pre challenged levelswithin 1.5 hrs.

Example 12 describes serum insulin quantification of bovine and rHinsulin on administering supramolecular insulin assembly (FIG. 13 a).Serum insulin was detectable up to a period of 150 and 180 days forbovine and rH insuline SIA-II, respectively, which corresponded to adecrease in BGL. The decrease in blood glucose levels was sustained upto a period of 150 and 180 days for bovine and rH insuline SIA-II,respectively, maintaining near normal glucose values for more than 20-25weeks with a single dose of the supramolecular insulin assembly II.Solid state ELISA was performed to quantify the plasma levels of insulinreleased from the insulin SIA II, which is responsible for themaintenance of normal blood glucose values. As expected, basal orslightly above basal level insulin release was achieved in case ofsupramolecular insulin assembly treated diabetic rats (0.5-1.2 ng/ml)compared to non-detectable insulin level (0.08 ng/ml) in PBS treateddiabetic rats (FIG. 13 a&c). A sustained release of insulin (0.8-1.1ng/ml) was observed from the day of injection up to 150-180 daysirrespective of the route, which contributed to near-normoglycemicvalues in the supramolecular insulin assembly treated animals. Whilesome variation was seen from animal to animal, remarkable values ofinsulin release could be achieved ranging from 0.5-1.2 ng/ml. This basallevel was enough to maintain normo-glycemic levels up until theexhaustion of the supramolecular insulin assembly. However, in insulintreated diabetic animals, a relatively higher mean serum level wasdetected, but with high degree of variation between animals. This wasattributable to inter-subject variability in BGL by insulin treatmentalone. Serum insulin levels were also quantified for the glucosetolerance test performed, for which glucose data has already been shown.FIG. 13 b, describes the bovine insulin values in the serum of rats upto 270 min in IPGTT experiments. In case of both control and STZ treatedrats given PBS only, the bovine insulin value is almost negligible (FIG.13 b). Whereas, in case of insulin administration, the insulin valuesincreases to ˜0.9 ng/ml in 30 min, and then decreases over a period oftime. Similar profile was observed when terminally released insulinwhich is equivalent to free insulin was used. This corresponds to thedecrease in blood glucose levels, followed by its increase due to uptakeand degradation of insulin. On the other hand, in supramolecular insulinassembly treatment, insulin levels in serum reached to 0.8-0.9 ng/ml,equivalent to the basal level in 30 min and then remained constant overthe period of study, as seen by bovine insulin quantification. Thissustained basal level ensures a normo-glycemic level, instead ofdecreasing the levels drastically and making the animal hypoglycemic.Further, Rat insulin ELISA was performed to evaluate the serum insulinlevels for both control rats and STZ treated ones. As shown in FIG. 13c, rat basal insulin levels is 0.501 ng/ml and increases manifold inresponse to infused glucose. In case of STZ treated rats, basal insulinlevels are almost negligible, due to the destruction of the pancreatic βcells by STZ. This confirms that the decrease in blood glucose levelsseen in case of supramolecular insulin assembly treatment is due to therelease of bovine/rH insulin released from the SIA II. In the process ofpreparation of insulin SIA II, insulin other than bovine and rH insulinselected from a group consisting of but not limited to pig insulin isemployed.

Example 13 provides I125 labeling of insulin to validate and quantifythe in vitro release from the termini of SIA II. Supramolecular insulinassembly II formed from labeled insulin has a specific activity of 49912CPM/ml/μg. 50 μl of supramolecular insulin assembly (4991200 CPM) wasinjected subcutaneously as well as intramuscularly and blood glucoselevels were monitored and serum samples were collected at 0, 30 min, 1h, 4 h, 10 h, 24 hrs, thereafter once a day, and then on alternate daysor once in a week. Counts in per ml of serum were measured (FIG. 14 a).As shown, blood glucose profile was same as observed with unlabeled SIAII. The CPM/ml calculated remained almost constant (FIG. 14 a) whenplotted against number of days of treatment. However there was aninitial high count at 30 min-4 hrs, which then gradually decreased toconstant level of 2000-3000 in 10 hrs. The amount of insulin released inblood was calculated and was in the range of 0.5-1.2 ng/ml whichcorresponds to the basal or slightly above basal level of insulin in theserum as observed with ELISA (FIG. 14 b). To further prove that releasedinsulin from supramolecular insulin assembly is monomeric, serum ofdifferent time points were resolved on tricine-SDS-PAGE and radiogramwas developed using the phosphor imager. As shown in FIG. 15, the bandin serum corresponds to free insulin monomer and its intensity remainedconstant for a long period when equal amount of serum was loaded. Adecrease in intensity was observed after 20 days showing usage anddepletion of the supramolecular insulin assembly depot over a period oftime together with some effect of the decay of the radio-label itself.

Example 14 describes hyperglycemic clamp studies performed to assess theslow and continuous release of insulin monomers from the injected SIAII. At one week, one month and three month time interval, the rate ofglucose infused for maintaining hyperglycemic state remained unchangedduring the course of the experiment. This shows that there is constantinsulin release from the depot at the site of injection. In case of thegroup being administered free insulin, the amount of glucose infuseddecreases over time, which is due to a decrease in the uptake of bloodglucose by muscle and liver in the absence of continuous supply of freeinsulin (FIG. 16 a-c).

Example 15 provides the effect of insulin on glucose transport and othermetabolic events in adipose tissues by performing primary culture of ratadiposites that are mediated by intracellular signaling cascades whichstart after insulin binds to insulin receptors on cell surface (PaulBevan Insulin signalling. J. Cell Sci., 114, 1429-1430 (2001)). Theeffect of insulin in the cell is mediated by the activation ofintracellular mediators like PI3K, AKT and ERK. Activation of the PI3kinase and Akt, mediates insulin induced glucose uptake and GLUT-4vesicle translocation to the plasma membrane and is involved in variousother insulin effects including inhibition of lipolysis and activationof fatty acid, glycogen, protein and DNA synthesis. On the other hand,activation of the extracellular signal-regulated kinase (ERK) pathway isimplicated in mitogenic responses of pre-adipocytes to growth factors.The level of PI3K which is the first kinase downstream to insulinsignaling was studied. As shown in FIG. 17 a, the level of PI3K isaugmented significantly when supramolecular insulin assembly and insulinreleased in vitro from them is used compared to control. There was nosignificant difference observed compared to insulin. The total proteinand activated level of Akt, a threonine/serine kinase important forregulation of insulin action and its various metabolic responses inadipocytes was also assessed. A significant increase in p-Akt level wasobserved which was again similar to the response observed with freeinsulin. There was no difference in total Akt protein level hence, nodifference in its expression in the presence of insulin or itssupramolecular insulin assembly II was observed. To assess whether theobserved temporal profiles and level of Akt phosphorylation inadipocytes was parallel to the difference in Akt activity, thephosphorylation of the endogenous Akt substrate GSK3β was monitored byimmunoblotting using antibodies to GSK3β and p-GSK3

GSK3

is directly phosphorylated by Akt on ser-21 and is inactivated followingits phosphorylation. The phosphorylation of GSK3

increased several fold in adipocytes treated with either free insulin orsupramolecular insulin assembly II compared to control and there was nosignificant difference among the treated cells. No difference wasobserved in total GSK3β protein level suggesting no change in itsexpression subsequent to treatment. Therefore, both Akt and GSK3

showed rapid and pronounced responses to supramolecular insulin assemblyII and released insulin from supramolecular insulin assembly and weresimilar to responses observed in stimulation with free insulin used as acontrol. The activation of ERK in treatment with supramolecular insulinassembly and insulin released from it in adipocytes was observed, asthis kinase is involved in insulin-mediated regulation of adipocytestranscription factors and adipose tissue development. On treatment ofadipocytes with insulin, supramolecular insulin assembly and releasedinsulin from supramolecular insulin assembly, a significant activationof ERK 1/2 was observed compared to control. The phosphorylation of ERK2 was two fold higher than ERK 1 suggesting more expression of ERK 2 ontreatment. There were no significant difference in phosphorylationpattern of ERK 1/2 in SIA, insulin or serum treated cells. Therefore theERK phosphorylation in supramolecular insulin assembly II and insulintreated cells were similar in the context of PI3K, Akt and GSK3

mediated signaling. Similar activation of signaling mediators wasobserved, when serum from insulin or its SIA-II treated animals wasadded to cultured adipocytes (FIG. 17 b). Together these datademonstrate that insulin released from supramolecular insulin assemblyII is able to activate the insulin signaling pathway in adipocytes asinsulin itself. The effects observed are even better as insulin in itsmonomeric form is released predominantly from its supramolecular insulinassembly.

Example 16 describes Western blot analyses of insulin, supramolecularinsulin assembly and in vitro released insulin (monomers) and serum fromrats treated with insulin, supramolecular insulin assembly and PBS.

Example 17 describes histology and immunohistochemistry details ofsupramolecular insulin assembly of insulin. The diabetic rats treatedwith supramolecular insulin assembly were monitored for the presence ofresidual amount of supramolecular insulin assembly from the day ofinjection from one week to 12 weeks. Supramolecular insulin assemblydeposits in subcutaneous as well as muscle tissues were detected usingCongo red. Subcutaneous and muscle tissue sections were prepared,stained and analyzed as described in materials and methods. Tissuesobtained after 24 h of supramolecular insulin assembly injection werefound to bind with Congo red effectively, confirming the presence ofhigher amounts of supramolecular insulin assembly fibrils (FIG. 18).Congo red binding decreased in a time dependent manner and wasnegligible after 16 weeks (FIG. 18 a(i-x) &b(i-x)), confirming therelease of insulin from the termini of supramolecular insulin assemblydeposits. Same tissues were also checked for inflammation caused bysupramolecular insulin assembly by H & E and immunostaining.Representative slides were subjected to H&E staining method andimmunohistochemistry for the presence of inflammatory cells (FIG. 18 a(xi-xx) and 18 b (xi-xv)).

As compared to the sections from LPS injected rats, where infiltrationof proinflammatory cells were visible in both subcutaneous and muscletissues stained with H &E (FIG. 19 a(i-ii)), supramolecular insulinassembly injected sections did not show any sign of inflammation evenafter 16 weeks (FIG. 18 a(xi-xv)). Similar results were observed in thecase of immunostained slides where LPS from Escherichia coli wassufficient to attract huge number of proinflammatory cells after 72 h atthe site of injection (FIG. 19 a (iii-iv)) whereas no such reaction wasobserved in animals treated with supramolecular insulin assembly (FIG.18 a (xv-xx) and b(xi-xv)). These observations reinforce non-cytotoxicnature of the supramolecular insulin assembly being used. Amyloid formedat pH 2.0 binds congo red dye very efficiently as seen in FIG. 19 b,when injected subcutaneously. Moreover there is no decrease in the depotcorroborating the data that no release of insulin monomers takes placefrom the fully formed amyloid (FIG. 19 b i-viii).

Example 18 describes alloxan model of diabetes. Male wistar rats wererendered diabetic using alloxan and their blood glucose levels weremonitored. Administration of supramolecular insulin assembly II todiabetic rats, lowered the blood glucose level to near normoglycemiclevels, and maintained a tight glycemic control for up to >120 days, inboth pre-prandial and post-prandial conditions (FIG. 20 a&b). Seruminsulin quantification was done using ELISA. From the day of injection,a sustained and constant release of human insulin is observed in theserum of the treated rats (0.5-0.9 ng/ml) (FIG. 20 c). Thus thephysiologic effect seen by the lowering of the glucose levels indiabetic rat is due to the continuous release of insulin monomers fromthe SIA-II depot formed at the site of injection.

Example 19 Reduction of secondary complications related to hyperglycemiain type I diabetes: In type-1 diabetes, insufficient supply of insulinleads to increased protein degradation in skeletal muscles and lipolysisin adipocytes. Diabetic rats showed a marked decrease in skeletal muscle(˜20-40%) and abdominal fat (>60%) (Nathan, D. M., Cleary, P. A.,Backlund, J. Y., et al. Intensive diabetes treatment and cardiovasculardisease in patients with type 1 diabetes. N. Engl. J. Med. 353, 2643-53(2005)). In contrast, supramolecular insulin assembly treated diabeticanimals had normal weight for these tissues and were healthy.Development of cataract in diabetic rats as well as in insulin treatedones were observed (FIG. 21). While no cataract formation was observedin animals treated with SIA II, majority of untreated rats exhibitedcataract as demonstrated in FIG. 21. Furthermore, the function of liverand kidney, the two main organs of the body which typically and severelyget afflicted in diabetes were normal in supramolecular insulin assemblyII treated rats as compared to their untreated or free insulin infusedrats and is summarized in Table 1.

Heart, kidney and liver sections were examined for the deposition ofhigher oligomers of SIA-II injected to diabetic rats after 12 weeks,visualized using congo red dye (FIG. 22). No deposition of oligomers wasobserved in any of the tissue sections.

Example 20 provides the details of detection of insulin degrading enzyme(IDE) and screening of antibodies against insulin. Subcutaneousresistance to insulin in Type-1 diabetes is a rare syndrome (Paulsen, E.P., Courtney, J. W. & Duckworth, W. C. Insulin resistance caused bymassive degradation of subcutaneous insulin. Diabetes 28, 640-645 (1979)and Freidenberg, G. R., White, N., Cataland, S., O'Dorisio, T. M.,Sotos, J. F. & Santiago, J. V. Diabetes responsive to intravenous butnot subcutaneous insulin: effectiveness of aprotinin, N. Engl. J. Med.305, 363-368 (1981)), defined as the lack of biological activity ofsubcutaneously injected insulin; nevertheless, efficacy forintravenously infused insulin is retained. This is mainly attributed toincreased insulin degradation by IDE in the subcutaneous tissues. IDEdegrades insulin specifically (Duckworth, W. C., Bennett, R. G. & Hamel,F. G. Insulin degradation: progress and potential, Endocr. Rev. 19,608-624 (1998)) and the partially degraded insulin is reabsorbed intothe circulation with increased immunogenicity but no biologicalactivity. IDE is present in insulin responsive tissues and in insulininsensitive cells such as monocytes and lymphocytes. To check thedevelopment of such a resistance in supramolecular insulin assemblytreated animals, biological activity of IDE and the presence ofanti-insulin antibody were determined as described in methods section.IDE activity was negligible in all the serum samples (1-12 weeks) fromsupramolecular insulin assembly treated animals (FIG. 23). Similarlythere was no presence of anti-insulin antibodies even after 12 weeks oftreatment, supporting the absence of IDE activity (FIG. 24).

To see whether the insulin monomers released from supramolecular insulinassembly II had undergone any change in its structure or its bindingdynamics, MTT assay on MCF 7 cell lines was performed. The proliferationof MCF 7 cells, when SIA was added to the culture was similar to that ofthose cells to which native insulin was added. Furthermore, the releasedmonomers from the SIA II formed, also showed the same kinetics of cellproliferation. Insulin like growth factor I was used as a positivecontrol for the proliferation of MCF 7 cells (FIG. 25). Insulin and IGF1 have similar structure, and in the absence of the other, each can bindto the other receptor. But whereas IGF 1 is mitogenic, insulin uponbinding to insulin receptor or IGF receptor does not cause proliferationof cells. Thus the monomers released from SIA II have the same bindingkinetics as that of native insulin, and do not undergo any structuralchange.

Example 21 describes Streptozotocin model for induction of diabetes inmice. Dose response for human and bovine SIA in mice rendered diabeticusing STZ. The administration of various dosage 20, 50, 100 and 200 μgof respective SIA to diabetic mice maintained the normo/near-normoglycemia for 5, 15, 30, 120 days respectively (FIG. 26 a-c).

Example 22 describes Streptozotocin model for induction of diabetes inrabbit. The administration of 1 mg/kg body weight of rH-insulin SIA-IIto diabetic rabbit maintained the normo/near-normo glycemia for atlest80 days (FIG. 27).

Example 23 provides details of the experiments carried out withsupramolecular insulin assembly along with Exendin 4. For treatment oftype II diabetes, supramolecular insulin assembly or exendin 4 wasadministered. The therapy administered was able to lower blood glucoselevels and maintain at near normoglycemic levels in diabetic rats upto >30 days (FIG. 28). The levels of TAGs and FFA also remained nearabout normal as compared to only insulin or PBS treated rats, where thelevels increased up to 1.5 fold in the serum as shown in Table 2.

The supramolecular insulin assembly formulation of the present inventionshows surprising result of release of insulin monomers for a longperiod. Further, the supramolecular insulin assembly II of the presentinvention does not exhibit an abrupt large release of insulin; thwartinghypoglycemic stage in STZ treated diabetic rats. To validate the aboveunexpected result the intermediates of insulin supramolecular assemblyII obtained during insulin fibrilization process at pH 7.0 was assessed.The intermediate supramolecular insulin assembly II is found to releaseinsulin monomers at a constant rate for long duration. The intermediate(supramolecular insulin assembly II) selected does not show significantCongo-red binding indicating its failure to progress to an amyloid stateand AFM analysis confirmed the presence of a linear association ofelongated oligomers as the predominant species. The height of thisunique entity is 18±5 nm compared to highly twisted fully grown fiberwith height 12±5 nm suggesting that the elongated oligomers which formsupramolecular insulin assembly are swollen and have retained nativelike structure. Similar non-fibrillar structures are also observed byTEM studies. The biological effectiveness of insulin is generallyassessed by its ability to regulate glycemic level in the blood. Theresultant decrease in the blood glucose concentration represents themost noticeable and, therapeutically, the most important effect ofinsulin. The efficiency of glycemic control in STZ-induced diabetic ratstreated with single dose of supramolecular insulin assembly and comparedwith single-daily insulin injections was assessed. Both supramolecularinsulin assembly treatment and insulin injection reduced the severity ofhyperglycemia. Compared to insulin treatment, single dose ofsupramolecular insulin assembly of the present invention maintainsnear-normoglycemic levels (120 mg/dL) in the diabetic animal for aperiod of about 150-180 days.

The present invention further assesses the effect of supramolecularinsulin assembly II therapy in regulation of blood glucose level. Thesupramolecular insulin assembly treated diabetic animals were subjectedto overnight fasting. These fasted animals were able to maintain theirfasting blood glucose levels within the normal range (60-100 mg/dl) andno pre-prandial hypoglycemia was observed. Taken together, these dataprovided that higher oligomeric state of supramolecular insulin assemblyII compared to conventional insulin therapy achieves a tightly regulatedglycemic control without fasting hypoglycemia in diabetic animals.

The present invention further assesses the effect of supramolecularinsulin assembly II treatment on body weight. Intraperitoneal glucosetolerance test conducted to determine the onset of the action ofsupramolecular insulin assembly in case of severe hyperglycemia. TheIPGTT blood glucose profile showed that both insulin and supramolecularinsulin assembly treatment showed their effect within 30 min, suggestingno lag phase in release of bioactive insulin monomers fromsupramolecular insulin assembly II depot.

The present invention provides in vivo release profile of insulin fromsupramolecular insulin assembly by bovine/human insulin quantificationin the serum using ELISA. In contrast to sigmoid release kineticsobserved in vitro, in vivo release of insulin monomers fromsupramolecular insulin assembly followed zero order kinetics as expectedfor a sustained release. A sustained release of basal and above basallevel (0.5-1.2 ng/ml) of insulin for maintaining normoglycemia was seen,which correlated well with the duration of the treatment. To validatethe above release kinetics, radiolabeling of insulin with 125I was doneand 125I labeled supramolecular insulin assembly were injected eithersubcutaneously or intramuscularly to STZ treated animals. The amount ofinsulin release was measured by monitoring CPM/ml. CPM/ml/μg (49912) wasused for the quantification of insulin released in the serum at aparticular time point and this paralleled to the amount of bovineinsulin quantified using ELISA. After measuring counts, serum sampleswere resolved on Tricine-SDS-PAGE. The phosphor images developed showedbands corresponding to insulin monomer. The insulin monomers releasedfrom the supramolecular insulin assembly depot triggered efficaciouslythe insulin signaling cascade in insulin responsive adipocytes. Insulinreleased from the supramolecular insulin assembly in vitro and in vivo(serum), when added to isolated adipocytes was able to activate theintracellular mediators (i.e. PI3K, Akt, ERK1/2 and GSK3β) of thesignaling pathway. Therefore, the insulin monomers released fromsupramolecular insulin assembly depot are biologically active and followthe same mechanism as insulin to regulate the glucose homeostasis in thebody. The histochemistry also provides that the supramolecular insulinassembly II injected in animals formed a depot from which there is aslow and sustained release of bioactive insulin. Furthermore, there isno inflammation observed as opposed to infiltration of leucocytes inresponse to subcutaneously or intramuscularly injected LPS.

The present invention provides that the supramolecular insulin assemblytherapy confers profound physiological benefits in diabetic animals.This is partially reflected in the significantly improved glycemiccontrol as well as markedly reduced urea and creatinine concentrationsdue to improved liver and kidney functions in diabetic rats as providedin Table 1.

The present invention provides details of anti-insulin antibodies andinsulin degrading enzyme (IDE) in serum on administration ofsupramolecular insulin assembly. The absence of anti-insulin antibodieseven by the end of the 12th week adds to the value of supramolecularinsulin assembly as a treatment for diabetes mellitus. Its inability toelicit IDE in or around the site of injection is an important factor inprolonging the release of insulin from the supramolecular insulinassembly and the concomitant beneficial anti-diabetic affect.

The present invention provides that insulin monomer released fromsupramolecular insulin assembly II has equivalent biological function assoluble insulin. A significant difference lies in the duration ofaction, whereas it is only the 6-10 hrs for standard insulin injection,but surprisingly so, about 10 days to about 180 days or more dependingon the dosage of the supramolecular insulin assembly II. This may beattributed to the remarkable stability of insulin in the supramolecularinsulin assembly, which forms a depot at the site of injection for thesupply of the most physiologically relevant form of insulin, viz.insulin monomers, for long periods of time.

Administration of supramolecular assembly II of human insulin led to aneven better glycemic control, observed over a period of 135-180 days.Both subcutaneous and intramuscular injection of the insulin oligomerresulted in near normoglycemia as evident from the FIGS. 8 b and 9 b.

Released insulin in serum was quantified using ELISA and was observed tomaintain an almost constant level, resulting from a slow and sustainedrelease from the depot.

Male wistar rats rendered diabetic with alloxan and streptozotocintreated diabetic C57b1/6 mice (served as another model of diabetes),both showed near normoglycemic blood glucose level upon being treatedwith supramolecular insulin assembly II.

The western blot data obtained for human supramolecular assembly IIinsulin is essentially the same, showing the activation of the signalingpathway by the released insulin monomers.

The supramolecular insulin assembly I is characterized by swollen andmore globular species with 18±2 nm height. The unfolding of the peptidestructure during the process of amyloid formation results in globularmonomeric species randomly distributed over the surface individually.Further onwards there is a linear association of these monomers at stageII of the human insulin supramolecular assembly II, albeit retainingtheir swollen morphology. The oligomers formed represent elongatedclusters, same as bovine insulin with a height of 18±4 nm. In case ofsupramolecular insulin assembly III (closer to the fibril stagesucceeding SIA II), an increase in density of higher oligomericstructures is seen. The structure is more compact with a height of 5±1nm. The overall structural morphology resembles bovine SIA stages, withfully formed fibers seen further onwards.

The present invention provides the efficacy as well as the feasibilityof supramolecular insulin assembly therapy for the significantimprovement of blood glucose level without causing fasting hypoglycemiain STZ-induced diabetic animal model. Unlike intensive insulin therapy,by which blood glucose levels is controlled by an increased frequency ofinsulin injection with concomitant risk of hypoglycemia, thesignificantly improved glycemic control using supramolecular insulinassembly therapy, is accomplished without the need for multiple insulininjections and without excessive body weight gain.

A further value to this innovation is added by the extension of thesestudies to those ailments requiring a sustained and continuous infusionof insulin as a therapy, as in the case of Diabetes Mellitus type II (DMII). Both Exendin 4 and insulin, in combination is utilized as a potenttherapy for DM II. Exendin 4 is known to lower blood glucose levels,through decrease in the absorption of food from the stomach. It alsodelays gastric emptying, lowers the levels of HbAc and prevents thelarge increase in weight observed due to insulin therapy. Exendin 4,aggregated into an oligomeric complex, from which the native monomers ofExendin 4 is released as is the case with insulin reported in the abovesection. Exendin 4 is used as an adjunct therapy along with SIA I and IIfor the treatment of DM II in animal subjects.

Example 24 describes monitoring of counter-regulatory hormones, whereinHyperinsulinemic Euglycemic/Hypoglycemic Clamp studies were performed tomonitor the levels of insulin counter-regulatory hormones, such asglucagon and epinephrine. A decrease of 40% was observed in the peaklevel of glucagon for SIA-II treated rats in comparison to normalcontrol Wistar rats (FIG. 29). In contrast, daily insulin treated ratsshowed 80-90% reduction in glucagon response. Thus, basal insulinrelease from SIA-II mimics the physiology of the body and does not causea significant alteration in the counter-regulatory mechanism of the bodyfor maintaining glucose homeostasis, unlike the daily insulin treatment.

Recurrence of hypoglycemia during SIA-II treatment in diabetic rats wasruled out by monitoring the insulin counter-regulatory hormone responseto induced hypoglycemia in various treated groups. Glycemic regulationthrough insulin counter-regulation was maintained in SIA-II treatedrats.

Example 25 describes protease resistance study to evaluate the stabilityof the supramolecular insulin assembly I, II and II.

Resistance of SIA-II and SIA-III to cleavage by Trypsin and Proteinase Kfurther demonstrate the difference in the structural organization ofthese oligomers. rH Insulin and SIA-I are more susceptible to cleavageby the proteases as compared to SIA-II and SIA-III (FIG. 30), whichadopt an higher order oligomeric form resistant to protease action.

Thus the present invention not only encompasses metabolic disease suchas Diabetes, both type I and II, but can be further extended to allthose diseases such as chronic pain, sepsis, arthritis, osteoporosis,inflammation, etc, where a continuous infusion of the therapeutic drugis required, be it a peptide, protein or a small drug molecule. Thisinvention also discusses the feasibility of using insulin oligomer (SIAI, SIA II and SIA III) for the treatment of DM I, DM II and borderlinediabetic cases. This methodology of utilizing the oligomers of the drugas a depot for treatment can be extended to many more diseases, having aall round, broad spectrum applicability.

The following examples are given by the way of illustration of theinvention contained in the present invention and therefore should not beconstrued to limit the scope of the present invention.

EXAMPLES

It should be understood that the following examples described herein arefor illustrative purposes only and that various modifications or changesin light of the specification will be suggestive to person skilled inthe art and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

Example 1 Insulin Fibrilization

Bovine and rH insulin, 2 mg/ml, was dissolved in phosphate buffer saline(50 mM, pH 7.0) or pH 2.0 (Hydrochloric acid in water) and incubated at37° C. for 72 hr-7 days with constant agitation at 180 rpm. The kineticsof fibril formation was monitored by monitoring the acquisition offluorescence by Thioflavin T (Th-T).

Example 2

Thioflavin T Fluorescence

Th-T fluorescence was measured on a Jobin Yvon Fluoromaxspectrofluorometer using slit widths of 3 nm and 5 nm for excitation andemission respectively. Samples incubated with 50 μM of Th-T for 15minutes were excited at 450 nm and their emission was monitored in therange of 460-560 nm. The data was corrected for blank and inner filtereffect using the following equation, Fc=F antilog[(A_(ex)+A_(em))/2]

Where Fc is the corrected fluorescence and F is the measured one, A_(ex)and A_(em) are the absorbance of the reaction solution at the excitationand emission wavelengths respectively.

Kinetics of formation of fibrils by bovine and rH insulin at 37° C. isprovided in FIG. 1. FIG. 1 (a) demonstrates kinetics of fibril formationat pH 7.0 monitored with 50 μM Th-T fluorescence.

Example 3 In Vitro Monomer Release Kinetics of Intermediates and FullyFormed Fibrils Formed at pH 2.0 and 7.0

200 μl (equal to 400 μg of insulin) aliquots were withdrawn at differenttime point from the insulin fibrilization reaction at pH 2.0 and 7.0,37° C. The supramolecular insulin assembly intermediate produced wereisolated by centrifugation. The pellet obtained was washed with PBS andresuspended in 1 ml PBS in an eppendorf. The cap of the eppendorfcontaining the intermediates was removed and the eppendorf was sealedwith 12 kDa cut off dialysis membrane. The eppendorf was then insertedthrough its membrane side into a 50 ml Falcon tube containing 20 ml PBSwith 0.02% sodium azide and the kinetics of the release of insulin wasmonitored for 15 days under constant stirring. The kinetics of releasewas monitored spectrophotometrically at 280 nm and by its intrinsic(tyrosine) fluorescence. The amount of insulin released per hour wascalculated. FIG. 2 a provides in vitro release of insulin fromSupramolecular insulin assembly II intermediate monitored by absorbanceat 280 nm and intrinsic tyrosine fluorescence. The Th-T intensity ofsolution inside the dialysis membrane at Oh and 15 days is also given.

To study release profile of different intermediates small aliquots ofintermediates and fully formed amyloid fibrils at both pH 2.0 and 7.0,37° C. were withdrawn from the fibrilizaton process at regular intervalsand centrifuged at 10,000 rpm for 10 minutes. Supernatant was removedand after washing the pellet twice with PBS, resuspended in fresh PBS.The release of monomeric insulin was monitored spectrophotometrically at280 nm, 37° C. Release kinetics was studied in two different conditions.In one, the pellet was suspended in PBS and dialyzed through a 12 kDacut off membrane in 20 ml PBS with constant stirring (FIG. 2 a-c). Insecond, the pellet obtained was suspended in 1 ml of PBS and theabsorbance of the supernatant was read at 280 nm (FIG. 2 d).

Example 4 Congo Red Binding

The amount of Congo red bound to the insulin oligomers/amyloid wasestimated as reported earlier (Klunk, W. E., Jacob, R. F. & Mason, R. P.Quantifying amyloid by Congo red spectral shift assay. Methods Enzymol309, 285-305 (1999)) using the equation, moles of Congo red bound/L ofamyloid suspension=A₅₄₀ nm/25295−A₄₇₇ nm/46306.

FIG. 3 provides congo-Red binding studies with native insulin,supramolecular insulin assembly II, supramolecular insulin assembly IIIand amyloid insulin.

Example 5 Tyrosine Fluorescence

The dialysate of supramolecular insulin assembly withdrawn at differenttime intervals in a 0.2 ml quartz cuvette was excited at 270 nm andemission recorded between 320 to 370 nm. Slit width of 5 nm was used forboth excitation and emission. FIG. 2( a) provides in vitro release ofinsulin from supramolecular insulin assembly II intermediate monitoredby absorbance at 280 nm and intrinsic tyrosine fluorescence. The Th-Tintensity of solution inside the dialysis membrane at Oh and 15 days isalso shown.

Example 6 Fourier Transform Infrared Spectroscopy (FTIR)

IR spectra were recorded with a Bruker Tensor 27 bench top FTIRspectrometer, equipped with a liquid N₂-cooled mercury cadmium telluridedetector. Insulin samples were analysed on Bio-ATR and 256interferograms were recorded at room temperature with a resolution of 2cm⁻¹. For each spectrum, water vapor was subtracted and baselinecorrected.

SIA I, II and III were also characterized using ATR-FTIR. Distinctspectra corresponding to each stage was observed (FIG. 4).

Supramolecular insulin assembly I, II and III of bovine and rH insulinwere also characterized using ATR-FTIR. Distinct spectra correspondingto each stage was observed (FIG. 4). A shift of the IR band towardslower frequencies is observed during fibrillization. Supramolecularinsulin assembly II (SIA-II) has a sharp peak at 1647 cm⁻¹ and 1645 cm⁻¹for bovine and rH insulin respectively, while the fully formed amyloidpeaks at 1630 cm⁻¹ and 1628 cm⁻¹ for the same. The FTIR spectra is ingood agreement with the CR binding data showing that the conformation ofSIA-II is still largely helical, albeit with an increase in the contentof random coil structure.

Example 7 Atomic Force Microscopy (AFM)

Pico plus atomic force microscope (Agilent Technologies) was used inmagnetic acoustic MAC (contact) mode for imaging. Images were recordedin air with either a bare mica surface or mica with sample using MACcantilever Type II (spring constant of cantilever: 2.8 N/m, Frequency:59.722 kHz). Samples were withdrawn from the fibrilization reactionmixture at various time points, diluted 20 fold with water andimmobilized on freshly cleaved mica for 2 minutes. The samples werewashed with nanopure water, dried under N₂ and subjected to AFManalysis.

FIG. 5 provides morphologies of supramolecular insulin assemblyintermediates and insulin fibrils studied by Atomic Force Microscopy.FIG. 5( a) insulin monomer (b) Supramolecular insulin assembly-Iintermediate, pH 7.0, (c) Supramolecular insulin assembly II, pH 7.0,(d) Supramolecular insulin assembly intermediate III, pH 7.0, (e) humanSIA I, (f) human SIA II, (g) human SIA III, and (h) Fully formed fibrilsat pH 7.0., (i) provide supramolecular insulin assembly intermediateformed at 6 hrs, pH 2.0 at 37° C., (j) provides fully formed amyloidfibril formed at pH 2.0.

Example 8 Transmission Electron Microscopy (TEM)

For TEM studies, samples were vortexed and immediately absorbed tofomber-coated 300 mesh copper grids as such or diluted to 1:2-20 foldwith mili-Q water and washed with deionized water. Grids were incubatedin 3% Uranyl Acetate for 2-5 min and dried under infra red light forexamining the samples by negative stain. The grids were visualized witha Phillips CM-10 at 80 kV. The pictures were captured using MegaView IIIcamera and analyzed using the Imaging Software from Imaging SystemPhillips. FIG. 6 provides negative staining TEM micrographs of insulinfibrils and supramolecular insulin assembly intermediates, wherein FIG.6( a) provides supramolecular insulin assembly I intermediate, pH 7.0,FIG. 6( b) provides supramolecular insulin assembly II, pH 7.0, FIG. 6(c) provides supramolecular insulin assembly intermediate III, pH 7.0,FIG. 6( d) provides mature fibers at pH 7.0, FIG. 6( e) provides fiberformed at pH 2.0, 37° C.

Example 9 Rat Model of Diabetes

Nine weeks old Male Wistar rats (Rattus norvegicus albinus, Rodentiamammalia) weighing 210±10 g were used. Rats were housed in commerciallyavailable polypropylene cages and maintained under controlledtemperature conditions on a 12 h light-dark cycle and allowed to accessfood and water ad libitum.

Streptozotocin Model for Induction of Diabetes in Rats

Male Wistar Rats weighing 250-300 g were divided into four groups andblood glucose estimation was done using Roche Accu Check glucose strips.Rats were kept on fasting for 48 hours. 50 mg/kg b·wt of Streptozotocinprepared freshly in citrate buffer (pH 4.5) was administeredintraperitoneally to 10-20 rats. Food was provided immediately and bloodglucose levels were checked after three days. The animals were groupedaccording to their blood glucose level (group I: 250-350 mg/dl, groupII: 350-450 mg/dl and group III: >450 mg/dL). High blood glucose levelswere maintained for a week with 2-6 U/kg body weight (b·wt) of bovineinsulin. All STZ-treated rats developed hyperglycemia (blood glucoselevels >250 mg/dl), 5 days after STZ injection, and their serum insulinlevels were quantified using rat insulin solid enzyme-linkedimmunosorbent assay (ELISA) (Mercodia). Rats with >250 mg/dL of glucoseand negligible (˜0.08 ng/ml) serum insulin levels were considereddiabetic and used for the experiment.

Example 10 Supramolecular Insulin Assembly Treatment

After one week of maintaining high blood glucose levels with insulin,the rats were divided into three groups, each containing 5 rats. Group Irats were administered single dose of 4 U/kg b·wt of bovine insulinintraperitoneally per day. Group II rats were injected 4 U/kg b·wt ofinsulin twice daily. Group III treated with 200 μg of supramolecularinsulin assembly II (subcutaneously as well as intramuscularly) in 100μl of PBS and group IV rats were administered 100 μl of PBS,constituting the diabetic control. A group of 5 normal rats injected 100μl of PBS, served as the non-diabetic control. Body weight and bloodglucose levels, both pre-prandial after 8-10 hr fasting andpost-prandial were checked initially daily and then with decreasingfrequency. FIG. 7 shows in vivo efficacy of supramolecular insulinassembly (alternatively pre-amyloid insulin) in glucose homeostasis. (a)Blood glucose level in response to various dosages of supramolecularinsulin assembly-II (bovine) administered both subcutaneously andintramuscularly. (b) Blood glucose level in response to various dosagesof supramolecular insulin assembly-II (r-human) administered bothsubcutaneously and intramuscularly.

FIG. 8 shows Post-prandial blood glucose levels monitored over a periodof 135 days after administration of bovine SIA-II (a) Bovine insulin,(b) rH insulin. FIG. 9 shows Pre-prandial blood glucose levels monitoredover a period of 160 days after administration of human SIA-II (a)Bovine insulin, (b) rH insulin. FIG. 10 shows blood glucose levelmonitored after administration of insulin amyloid formed at pH 2.0 and7.0. FIG. 11 shows body weight profile of SIA-II treated diabetic rats,diabetic control and non-diabetic control rats.

Example 11 Intraperitoneal Glucose Tolerance Test (IPGTT)

The STZ treated (n=12) and normal rats (n=4) were kept on fasting for 12hrs. Blood glucose levels were monitored as described above. For glucosetolerance test was done. Briefly, animals were infused with 3 g/kg bodyweight of glucose, intraperitonealy, followed by injection of 4 U/kgb·wt of Bovine insulin to group I, 100 μl of amyloid insulin to group IIand 100 μl of PBS (vehicle) to group III rats. Blood glucose levels weremonitored at 0, 30, 90, 150, 270 and 330 min after treatment. Serum wasisolated for various time points and a graph was plotted between bloodglucose level and time. FIG. 12 provides blood glucose profile ofIntraperitoneal Glucose Tolerance Test (IPGTT).

Example 12 Serum Insulin Quantification

Serum was isolated from the blood samples collected and stored at −20°C. till further analysis. Bovine and rat insulin levels were quantifiedusing solid phase two site enzyme immunoassay (ELISA) from Mercodia(Sweden), by following the manufacturer's protocol. FIG. 13 a providesquantification of serum human and bovine insulin using ELISA in STZtreated rats in response to supramolecular insulin assembly injected SCor IM, FIG. 13( b) provides quantification of serum bovine insulin ofIPGTT, FIG. 13( c) provides serum rat insulin ELISA performed for IPGTT.

Example 13 I¹²⁵ Labelling of Insulin

To further validate and quantitate the in vitro release from the terminiof insulin SIA II, labeling of insulin with ¹²⁵I was done (Pause, E.,Bormer, O. & Nustad, K. Radioiodination of proteins with the iodogenmethod, in RIA and related procedures in medicine, international atomicagency, Vienna, 161-171 (1982)). Supramolecular insulin assembly formedfrom labeled insulin had a specific activity of 49912 CPM/ml/μg. 50 μlof supramolecular insulin assembly (4991200 CPM) was injected eithersubcutaneously or intramuscularly and blood glucose levels weremonitored and serum samples were collected at 0, 30 min, 1 h, 4 h, 10 h,24 hrs, thereafter once a day, and then on alternate days or once in aweek. Counts in per ml of serum were measured (FIG. 14 a). As shown inFIG. 14 b, blood glucose profile was same as observed with the unlabeledSIA II. The CPM/ml calculated remained almost constant (FIG. 14 a) whenplotted against the number of days of treatment. However, there was aninitial high count at 30 min-4 hrs, which then gradually decreased to aconstant level of 2000-3000 in 10 hrs. The amount of insulin released inblood was calculated and was in the range of 0.5-1.2 ng/ml whichcorresponded to the basal or slightly above basal level of insulin inthe serum as observed with ELISA (FIG. 14 b). To further prove that thereleased insulin from supramolecular insulin assembly is monomeric,serum of different time points were resolved on tricine-SDS-PAGE(Schaogger, H. & Von Jagow, G. Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa. Anal Biochem 166, 368-379(1987)) and radiogram was developed using the phosphor imager. As shownin FIG. 15, the band in serum corresponds to free insulin monomer andits intensity remained constant for a long period when equal amount ofserum was loaded. A decrease in intensity was observed after 20 daysshowing usage and depletion of the supramolecular insulin assembly depotover a period of time together with some effect of the decay of theradio-label itself.

Example 14 Hyperglycemic Clamp after Treatment with SupramolecularInsulin Assembly II

Male wistar rats were anesthetized (Isoflurane 2%) and a carotid andjugular catheter installed to allow blood withdrawal and glucoseinjections (20% glucose solution) to clamp blood glucose level at 600mg/dL. Following a 12 hours fasting period, glucose was infused in allgroups to make them hyperglycemic. This was followed by blood withdrawalat indicated time intervals, for blood glucose measurements and the rateof glucose infusion to retain them hyperglycemic were calculated. Thisprocedure was repeated after one and three months of SIA IIadministration (FIG. 16 a-c).

Example 15 Isolation and Primary Culture of Rat Adipocytes

Rat adipocytes were isolated and cultured according to the methoddescribed by Bjorntorp et al. (Bjorntorp, P., Karlsson, M., Pettersson,P. & Sypniewska, G. Differentiation and function of rat adipocyteprecursor cells in primary culture. J. Lipid Res. 21, 714-723 (1987)).Male Wistar rats fed freely were sacrificed and epididymal fat tissuewas dissected and collected in reagent A (HBSS, 100 U/ml penicillin, 100μg/ml streptomycin and 50 μg/L gentamycin). The tissue was washedproperly in HBSS. Following this the tissue was cut and minced finelyand transferred to a falcon, centrifuged at 200 g for 2 min. The layerof transparent oil was removed and the adipocyte cell layer (thick anddense) was added to three times the volume of reagent B (reagent Acontaining 0.1% BSA and 1 mg/ml Collagenase) in a flask. The flask wasincubated at 37° C. for 60 min with continuous slow shaking. Thereaction was stopped by addition of DMEM complete media (with HEPES 15mM, glc, 0.1% BSA, 50 nM adenosine and 1% fetal bovine serum) threetimes the volume and incubated at room temperature for 5 min. Thereaction was transferred to a falcon and centrifuged at 200 g for 10min. Adipocytes were collected after discarding the top layer of oil andwashed twice with reagent A by centrifuging at 200 g for 10 min. Cellswere dispensed into a flask with appropriate volume of DMEM completemedia and incubated for 24 hrs at 37° C. For insulin signaling,adipocytes were centrifuged and maintained in serum free medium for 12hrs before plating approximately 2 ml into 6 well culture plates, andincubated further for 2 hrs.

Example 16 Western Blot Analysis of Total Cellular Lysates

Plated cell were incubated with either 20 nM insulin, 50 μl ofsupramolecular insulin assembly and in vitro released insulin (monomers)or 50 μl of serum from rats treated with insulin, supramolecular insulinassembly and PBS for 10 min. After incubation, the cells were collectedin a falcon tube and centrifuged at 200 g for 10 min. The top layer ofadipocytes were collected in an eppendorf and kept in ice. 500 μl oflysis buffer (20 mM Tris pH 8.0, 1% NP 40, 137 mM NaCl, 1 mM MgCl₂, 1 mMCaCl2, 1 mM DTT, 10% glycerol, 1 mM PMSF, 0.4 mM sodium orthovanadateand protease inhibitor cocktail) was added and the samples were frozenat −80° C., for 2 hrs. This was followed by thawing and incubating at 4°C. for 4 hrs with constant rotation. The supernatant was collected aftercentrifugation at 13000 rpm for 30 min and protein concentration in celllysate was estimated using Bradford reagent. Fifty micrograms of totalcellular protein were applied to each lane and were separated on 10%SDS-PAGE and transferred to nitrocellulose membrane using Bio-rad wettransfer apparatus at 4° C. overnight. After transfer, the blot wasremoved and stained with Ponceau-S for the visualization of transferredbands, and destained further with water. The membrane was blocked for 1hr at 37° C. with 5% skimmed milk in PBS, pH 7.4, washed and thenincubated overnight in primary antibody (1:1000 dilution using 1%skimmed milk in PBS) of PI3K, p-Akt, total Akt, p-Gsk3β, Gsk3β, ERK1/2,GAPDH and β actin (antibodies from cell signaling) at 4° C. Afterwashing with PBST, the blot was incubated for 1 hr in respectivesecondary antibody (HRP-conjugated), and immunoreactive bands werevisualized using the ECL western blotting protocol (Amersham). FIG. 17provides western blot (WB) analysis of cultured adipocytes for insulinsignaling cascade. Adipocytes treated with (a) PBS, insulin, SIA-II,insulin released from SIA-II, (b) serum as indicated, and analysed forinsulin signaling.

Example 17 Histology and Immunohistochemistry

Rats were injected 200 μg of insulin SIA-II or 150 μg ofLipopolysaccharides (LPS) from Escherichia coli (Sigma-Aldrich, MO, USA)either through intramuscular or subcutaneous injections into thighmuscle and dorsal skin respectively. LPS injected rats were sacrificedafter 48 h of injection whereas rats injected insulin SIA-II weremonitored from 1 to 12 week and tissue sections were excised at aninterval of every 7 days. Rats were anesthetized by ketamine andperfused with 4% paraformaldehyde. Skin and thigh muscles were removedand the injection site was excised out. Tissues were then processed forparaffin embedding and were sagitally sectioned at a thickness of 10 μmand further processed for routine hematoxylin-eosin (H & E) staining tosee histology and the infiltration of inflammatory cells, Congo redstaining (Lee, G. & Luna, H. T. Manual of Histologic staining methods ofarmed forces institute of pathology. 3rd Ed. McRaw-Hill book company(1960)) for the presence of residual SIA-II and Immunohistochemistry(Sanz M J, Marinova-Mutafchiev L, Green P, Lobb R R, & Feldmann M,Nourshargh S. IL-4-induced eosinophil accumulation in rat skin isdependent on endogenous TNF-alpha and alpha 4 integrinNCAM-1 adhesionpathways. J Immunol. 160, 5637-5645 (1998)) with antibodies againstCD11b, RT-1A, and CD6 (BD Pharmigen, CA, USA). All immunoflorescentslides were mounted permanently with antifade reagent+mounting medium(Molecular probes, Eugene, Oreg., USA) and observed under florescentlight for FITC conjugated antibodies. CR and H&E stained slides weremounted with citramount medium (Polysciences, PA, USA). H&E sectionswere observed under bright light whereas, CR stained slides under brightand polarized lights (Nikon Eclipse 80i, Nikon, Japan). Images werecaptured using DS SMc CCD camera (Nikon, Japan) and were analyzed byNIS-Element software (Nikon, Japan).

Example 18 Alloxan Model for Induction of Diabetes in Rats

Male Wistar Rats weighing 250-300 g were divided into four groups andblood glucose estimation was done using Roche Accu Check glucose strips.Rats were kept on fasting for 24 hours. 150 mg/kg b·wt of Alloxanprepared freshly in citrate buffer (pH 4.5) was administeredintraperitoneally to 10-20 rats. Food was provided immediately and bloodglucose levels were checked after three days. The animals were groupedaccording to their blood glucose level (group I: 250-350 mg/dl, groupII: 350-450 mg/dl and group III: >450 mg/dL). High blood glucose levelswere maintained for a week with 2-6 U/kg body weight (b·wt) of bovineinsulin. 60% of Alloxan-treated rats developed hyperglycemia (bloodglucose levels >250 mg/dl), 5 days after injection, and their seruminsulin levels were quantified using rat insulin solid enzyme-linkedimmunosorbent assay (ELISA) (Mercodia). Rats with >250 mg/dL of glucoseand negligible (˜0.18 ng/ml) serum insulin levels were considereddiabetic and used for the experiment.

Example 19 Examples of Clinical Parameters Examined

Biochemical assays were performed for the evaluation of toxicity ofsupramolecular insulin assembly treatment. Serum glutamate oxalo-acetatetransaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), totalBilirubin, Bilirubin, Alkaline Phosphatase, Serum total proteins, SerumAlbumin, Serum Globulin, Serum A/G ratio, Kidney function test (KFT),Cataract Formation, Adipose Tissue weight, Body Weight and Appearancewere estimated using assay kits available from Merck India Ltd. Table 1provides the analysis of clinical parameters for the evaluation oftoxicity of Insulin SIA II. Serum isolated from blood samples collectedat the end of the three month study and subjected to various testsindicated in the Table. Results are mean±s.d. of three differentexperiments having n=4 animals in each group.

Example 20 Detection of Anti-Insulin Antibodies and Insulin DegradingEnzyme (IDE) in Serum

Indirect ELISA was performed for the detection of anti-insulinantibodies in the rat serum by following the standard ELISA protocol.Indirect ELISA was performed for the detection of anti-insulinantibodies in the rat serum by following the standard ELISA protocol.Briefly, 200 μl of 2 mg/ml of bovine insulin in 50 mM carbonate buffer,pH 9.6, was coated onto a 96 well ELISA plate and kept overnight at 4°C. 5% BSA in PBS was used for blocking at 37° C. for 1 hr. The plate wasthen washed with PBST (0.02% Tween 20) and 20031 of 1:100 diluted serumwas added and kept at 37° C. for 1 hr. Further rounds of washing withPBST was followed by the addition of 1:10000 diluted anti-rat IgG-HRPconjugated 2° antibody and incubated for 2 hrs, 37° C. Color wasdeveloped using TMB as a substrate and reaction stopped by the additionof conc H₂SO₄. The plate was read at 450 nm spectrophotometrically.Anti-insulin antibody was used as a positive control for the reaction.IDE was quantified from the serum using Insulysin/IDE InnoZyme™Immunocapture Activity Assay Kit (Calbiochem) following themanufacturer's protocol. Rat IDE provided in the kit served as thepositive control.

Cell Proliferation Assays

Cells were plated in 24-well plates with 10,000 cells/well in regularDMEM media containing 10% FBS. Cells were switched to Serum Free Mediafor 12 h and then treated as indicated in the figure legends of FIG. 25.All treatments were done in triplicates. Growth was measured 3 daysafter treatment. Growth was assayed by the MTT assay. A total of 50 μlof 5 mg/ml MTT solution in PBS was added to each well. After incubationfor 4 h at 37° C., formazan crystals were lysed with 500 μl ofsolubilization solution (20% SDS, 50% DMSO). Absorbance was measuredwith a plate reader at 570 nm using a 670-nm differential filter.

Example 21 Streptozotocin Model for Induction of Diabetes in Mice

Inbred 12-16-week-old C57BL/6 male mice were used. Mice were injectedintraperitoneally with 50 mg/kg b·wt of streptozotocin daily for 5 days.Blood glucose levels were estimated after two weeks. Mice with >300mg/dl of blood glucose were considered diabetic and selected for furtherexperiments. A total of six groups were made, with each group consistingof six mice each. Various dosages, such as 10 μl, 25 μl, 50 μl and 100μl of bovine/human insulin SIA-II was administered either subcutaneouslyor intramuscularly to mice rendered diabetic using Streptozotocin, withthe two other groups serving as the diabetic and the non-diabetic group.Both fasting and fed blood glucose levels were monitore using the RocheAccu Check glucose strips.

Example 22 Streptozotocin Model for Induction of Diabetes in Rabbit

Male New Zealand rabbits, weighing between 100 μl and 1200 g were used.Animals were maintained under controlled conditions of humidity,temperature (22±2° C.) and 12 h light and dark cycle. The experimentalprotocol and animal handling was in accordance with the Institutionalanimal ethics committee of the National Institute of Immunology, NewDelhi, India. For induction of experimental diabetes, rabbits used werefasted for 12 h, followed by administration of 80 mg/kg b·wt ofStreptozotocin, prepared in citrate buffer, pH 4.5. Blood glucose levelswere checked after three days. Rabbits showing BGL >450 mg/dL weretermed diabetic and further divided three groups of three rabbits each.Group I—normal healthy rabbits, group II—diabetic treated with insulin,group III—diabetic treated with SIA-II (SC) and group IV—diabetictreated with PBS.

Example 23 Model for Induction of Diabetes Type II in Wistar Rats andits Treatment Using SIA

Male Wistar rats, 7 weeks of age, and weighing approximately 200 g, wereused for all studies. Animals were fed either a normal chow dietconsisting (as a percentage of total kcal) of 12% fat, 60% carbohydrate,and 28% protein or a high-fat diet consisting of 40% fat, 41%carbohydrate, and 18% protein. After 2 weeks on either diet, animals(with the exception of non-injected controls) after an overnight fastwere injected with STZ (50 mg/kg) into the tail vein via a temporaryindwelling 24-gauge catheter. Animals had free access to food and waterafter the STZ injection, and both STZ-injected and non-injected animalswere continued on their original diets (chow or fat) for the duration ofthe study. Animals with high blood glucose levels were administeredeither PBS as vehicle, insulin SIA, or insulin SIA with Exendin 4asubcutaneously. Blood was collected and serum was separated bycentrifugation and analyzed for concentrations of glucose (glucosestrips, Accucheck, Roche), insulin (Insulin Elisa Kit, Mercodia),triglyceride (TG) (glycerol phosphate oxidase [GPO]-Trinder method,Sigma) and free fatty acid (acyl coenzyme A synthetase [ACS-ACOD]method, Wako Diagnostics, Richmond, Va.).

Diabetes Type II: Db/db Model

Db/db mice on a C57BL/6 background were fed ad libitum with a standarddiet and kept under a 12-h light/dark cycle. Blood samples werecollected via mouse tail bleeds, and circulating glucose levels weredetermined using a glucometer (Roche Accu Check glucose strips). Seruminsulin levels were determined from serum samples using an insulinenzyme linked immunosorbent assay kit (Mercodia). Fasting blood glucoseand random-fed glucose were performed in the morning on alternate days.

Example 24 Insulin Counter-Regulatory Hormone Monitoring

The hyperinsulinemic glucose-clamp procedure was followed to provide afixed hypoglycemic stimulus to rats. Animals were catheterized asdescribed above. Conscious and unstressed rats were fasted for 12-14hours before the start of the experiment. Constant insulin infusion of30 mU/kg·min was begun along with a variable infusion of exogenousglucose, which was adjusted based on the blood glucose measurementobtained at 10 min intervals to achieve the desired glucose level.During the first 90 min of the experiment, the rats were brought toeuglycemia, ˜110 mg/dL. Thereafter, blood glucose level was decreased to˜50 mg/dL (induced hypoglycemia by infusion of insulin), and wasmaintained for the next 90 min. Experiments were terminated if theglucose levels fell below 80 mg/dL and 40 mg/dL during the euglycemicand the hypoglycemic phase, respectively. Blood samples for measurementof glucagon and epinephrine were withdrawn at various time intervals, asindicated in the FIG. 29.

Example 25 Protease Resistance

To 20 μl of 2 mg/ml rH Insulin, SIA-I, SIA-II and SIA-III, 1:5, 1:10 and1:50 dilution of 2 mg/ml trypsin and 1:1000, 1:2000 and 1:5000 dilutionof 2 mg/ml Proteinase K was added. The reaction mixture was incubatedfor 12 hrs at 37° C. in an incubator. The samples were loaded onto 20%SDS-PAGE and analyzed using Coomassie stain.

TABLE 1 Analysis of clinical parameters for the evaluation of toxicityof Supramolecular Insulin Assembly II (SIA II) Single daily InsulinTwice daily Parameters estimated Normal rats SIA II treated InjectionInsulin Injection LFT Bilirubin (Total) (mg/dL)  0.35 ± 0.03  0.35 ±0.06  0.40 ± 0.08* 0.35 ± 0.1 Bilirubin (Direct) (mg/dL) 0.108 ± 0.02 0.1 ± 0.03  0.36 ± 0.08* 0.25 ± 0.06* SGOT (U/L) 222.8 ± 79 219.8 ± 61  355 ± 83*  300 ± 56* SGPT (U/L)  74.8 ± 17   77 ± 21   86 ± 36   85 ±15 Alkaline Phosphatase (U/L) 343.2 ± 76.8   431 ± 70.3 777.8 ± 89.4* 489 ± 65 Serum total proteins (g/dL)  3.92 ± 0.095  6.14 ± 0.3  6.70 ±0.21* 5.55 ± 0.25* Serum Albumin (g/dL)  1.77 ± 0.11  1.85 ± 0.13  2.5 ±0.21  3.2 ± 0.19 Serum Globulin (g/dL)  2.15 ± 0.08  2.64 ± 0.12  4.2 ±0.18 3.35 ± 0.15 Serum A/G ratio 0.823 1.3 0.595 0.955 KFT Urea (mg/dL)47.68 ± 10.8  50.9 ± 7.6  58.3 ± 9.2 60.1 ± 12.36 Serum Creatinine(mg/dL)  0.80 ± 0.06  0.83 ± 0.02  1.01 ± 0.30 0.88 ± 0.21 Uric Acid 2.39 ± 0.12  2.26 ± 0.08  3.0 ± 0.81  3.1 ± 0.56 Electrolytes Sodium(mEq/L) 140.6 ± 11   144 ± 10   200 ± 26  198 ± 45.5 Phosphorous (mEq/L) 4.5 ± 1.01  4.4 ± 1.1  6.5 ± 1.3  6.2 ± 0.95 Chloride (mEq/L) 102.8 ±12   101 ± 17   165 ± 23  119 ± 26 Cataract Formation (−) (−) (+) (+)Adipose Tissue Normal Normal Decreased Decreased Body Weight andAppearance (+++) (+++) (+) (++)

TABLE 2 Effects of Treatments on the metabolic parameters of blood infat-fed/streptozotocin-diabetic rats 1 week 2 week Rat Rat BGL InsulinFFA TG BGL Insulin FFA TG Group (mg/dL) (μU/ml) (mmol/L) (mmol/L) B W(mg/dL) (μU/ml) (mmol/L) (mmol/L) B W Control 107  20.5 ± 1.95 0.87 ±0.10 0.48 ± 0.17 253 98 19.5 ± 1.5 0.89 ± 0.10 0.49 ± 0.17 260 Fat 47739.48 ± 6.72 2.45 ± 0.07 0.86 ± 0.04 265 480  35.38 ± 10.18 2.55 ± 0.070.89 ± 0.04 271 fed/STZ Control Fat 420 37.09 ± 4.7   1.9 ± 0.08 0.77 ±0.05 261 401 37.09 ± 4.7   2.1 ± 0.08 0.75 ± 0.05 263 fed/STZ + InsulinFat 180 34.83 ± 5.96  1.1 ± 0.06 0.68 ± 0.12 264 158 36.96 ± 5.04  1.0 ±0.06 0.68 ± 0.12 268 fed/STZ + Insulin SIA Fat 157 31.58 ± 6.75 0.91 ±0.11 0.51 ± 0.13 252 121 21.58 ± 6.75 0.88 ± 0.11 0.50 ± 0.13 259fed/STZ + Exendin- 4 SA 3 week 4 week Rat Rat BGL Insulin FFA TG BGLInsulin FFA TG Group (mg/dL) (μU/ml) (mmol/L) (mmol/L) B W (mg/dL)(μU/ml) (mmol/L) (mmol/L) B W Control 100  20.1 ± 0.95 0.86 ± 0.10 0.51± 0.17 265 98  20.5 ± 1.95  0.9 ± 0.10 0.47 ± 0.17 276 Fat 506 39.55 ±6.3  2.56 ± 0.07 0.91 ± 0.04 256 511 40.15 ± 1.35 2.59 ± 0.07 0.94 ±0.04 250 fed/STZ Control Fat 390 37.09 ± 4.7  1.8 ± 0.08 0.73 ± 0.05 269396 37.09 ± 4.7   2.1 ± 0.08 0.76 ± 0.05 272 fed/STZ + Insulin Fat 14936.96 ± 5.04  0.9 ± 0.06 0.68 ± 0.12 272 163 22.42 ± 8.70  1.1 ± 0.060.68 ± 0.12 276 fed/STZ + Insulin SIA Fat 119 19.40 ± 4.86 0.87 ± 0.110.48 ± 0.13 265 158 23.40 ± 4.86 0.98 ± 0.11 0.65 ± 0.13 272 fed/STZ +Exendin- 4 SA

What is claimed is:
 1. A supramolecular insulin assembly (SIA) useful asprotein therapeutics for the treatment of metabolic disorders selectedfrom the group consisting of type 1, type 2 diabetes mellitus andcomplications thereof, wherein said assembly comprises insoluble andaggregated oligomeric form of insulin.
 2. The supramolecular insulinassembly (SIA) as claimed in claim 1, wherein said assembly comprisesinsoluble and aggregated oligomeric form of insulin as SIA I, SIA II,SIA III or combination thereof, wherein SIA I consists of elongatedclusters having pearl like arrangement of insulin monomer, SIA IIconsists of linear association of elongated clusters having pearl likearrangement of insulin monomer and SIA III consists of about 90% SIA-II,that is a dense, linear association of insulin oligomers.
 3. Thesupramolecular insulin assembly (SIA) as claimed in claim 1, whereinsaid assembly comprises insoluble and aggregated oligomeric form ofinsulin as SIA I, wherein SIA I consists of elongated clusters havingpearl like arrangement of insulin monomer.
 4. The supramolecular insulinassembly (SIA) as claimed in claim 1, wherein said assembly comprisesinsoluble and aggregated oligomeric form of insulin as SIA II, whereinSIA II consists of linear association of elongated clusters having pearllike arrangement of insulin monomer.
 5. The supramolecular insulinassembly (SIA) as claimed in claim 1, wherein said assembly comprisesinsoluble and aggregated oligomeric form of insulin as SIA III, whereinSIA III consists dense, linear association of insulin oligomers.
 6. Thesupramolecular insulin assembly (SIA) as claimed in claim 1, whereinsaid insulin is recombinant human insulin, bovine or pig insulin, ormutants/analogs of insulin.
 7. The supramolecular insulin assembly (SIA)as claimed in claim 1 or 2, wherein said assembly releases insulin at arate ranging from 0.1 to 5.4 ng/ml, wherein rate of release of insulinis in the range of for about 7 to 180 days, in vivo.
 8. Thesupramolecular insulin assembly (SIA) as claimed in claim 3, whereinsaid assembly releases insulin at a rate ranging from 4-5.4 ng/ml for atleast 7-10 days.
 9. The supramolecular insulin assembly (SIA) as claimedin claim 4, wherein said assembly releases insulin at a rate rangingfrom 0.5-1.8 ng/ml for at least 160 days.
 10. The supramolecular insulinassembly (SIA) as claimed in claim 5, wherein said assembly releasesinsulin at a rate ranging from 0.1-0.7 ng/ml for at least 180 days. 11.The supramolecular insulin assembly (SIA) as claimed in claim 4, whereinsaid assembly upon administration to diabetic subjects maintainsnear-normoglycemic level (120±30 mg/dl) for at least 160 days in asubject in need thereof.
 12. The supramolecular insulin assembly (SIA)as claimed claim 1, wherein a single dose of said assembly uponadministration maintains near-normoglycemic level (120±30 mg/dl) for atleast 7 to 180 days in a subject in need thereof, wherein concentrationof said assembly in said dose is in the range of 0.125 to 3.75 mg/kgbody weight.
 13. The supramolecular insulin assembly (SIA) as claimed inclaim 1, wherein a single dose of said assembly upon administrationmaintains near-normoglycemic level (120±30 mg/dl) for at least 160 daysin a subject in need thereof, wherein concentration of said assembly insaid dose is in the range of 0.75 to 1.25 mg/kg body weight.
 14. Asupramolecular exendin-4 assembly (SEA) useful as protein therapeuticsfor the treatment of diabetes, wherein said assembly comprises insolubleand aggregated oligomeric form of exendin-4.
 15. The supramolecularinsulin assembly as claimed in claim 1 is a non cytotoxic, nonimmunogenic, non-apoptotic and non-mitogenic prodrug.
 16. Thesupramolecular exendin-4 assembly as claimed in claim 14 is a noncytotoxic, non immunogenic, non-apoptotic and non-mitogenic prodrug. 17.A pharmaceutical composition for the treatment of metabolic disordersselected from the group consisting of type 1, type 2 diabetes mellitusand complications thereof, said composition comprising therapeuticallyeffective amount of the supramolecular insulin assembly (SIA) as claimedin any one of claims 1, 2, 3, 4 or
 5. 18. A pharmaceutical compositionfor the treatment of metabolic disorders selected from the groupconsisting of type 1, type 2 diabetes mellitus and complicationsthereof, said composition comprising therapeutically effective amount ofthe supramolecular insulin assembly (SIA) as claimed in any one ofclaims 1, 2, 3, 4 or 5 and supramolecular exendin-4 assembly (SEA) asclaimed in claim
 14. 19. The pharmaceutical composition as claimed inany of the claim 17 further comprises pharmaceutically acceptablecarriers, additives or diluents.
 20. The pharmaceutical composition asclaimed in any of the claim 18 further comprises pharmaceuticallyacceptable carriers, additives or diluents.
 21. The pharmaceuticalcomposition as claimed in any of the claim 17 is administeredintramuscularly, intradermally or subcutaneously.
 22. The pharmaceuticalcomposition as claimed in any of the claim 18 is administeredintramuscularly, intradermally or subcutaneously.
 23. The pharmaceuticalcomposition as claimed in claim 17 is administered through a devicecapable of releasing the said composition, wherein said device isselected from a group consisting of pumps, catheters, patches andimplants.
 24. The pharmaceutical composition as claimed in claim 18 isadministered through a device capable of releasing the said composition,wherein said device is selected from a group consisting of pumps,catheters, patches and implants
 25. A process of preparation ofsupramolecular insulin assembly (SIA) as claimed in claim 1, saidprocess comprising; a. dissolving insulin at a temperature of about 25to 60° C. in a solution having pH in the range of about 1.5 to 7.8; andb. incubating the above for a period of about 6 to 48 hours withconstant shaking to obtain Supramolecular Insulin Assembly (SIA),wherein SIA comprises insoluble and aggregated oligomeric form ofinsulin
 26. The process as claimed in claim 25, wherein said processfurther comprises a washing said SIA with PBS; and b re-suspending saidSIA in PBS
 27. The process as claimed in claim 25, wherein said periodis about 8 to 12 hours preferably 10 hours.
 28. A process of preparationof supramolecular exendin-4 assembly as claimed in claim 14, saidprocess comprising; a. dissolving exendin-4 at a temperature of about 25to 60° C. in a solution having pH in the range of about 2.0 to 7.6; andb. incubating the above for a period of 6 to 192 hours with constantshaking to obtain Supramolecular Exendin-4 Assembly (SEA), wherein SEAcomprises insoluble and aggregated oligomeric form of Exendin-4
 29. Theprocess as claimed in claim 28, wherein said process further comprisesa. washing said SEA with PBS; and b. re-suspending said SEA in PBS 30.The process as claimed in claim 28, wherein said period is 148 hours.31. The process as claimed in claim 25, wherein the solution is selectedfrom hydrochloric or acetic acid in water having pH in the range ofabout 1.5 to 2.5; sodium acetate buffer having pH in the range of about3.5 to 5.5; phosphate buffer (PBS) having pH 6 to 7.8, and citratebuffer having pH in the range of about 4 to
 6. 32. The process asclaimed in claim 28, wherein the solution is selected hydrochloric oracetic acid in water having pH in the range of about 1.5 to 2.5; sodiumacetate buffer having pH in the range of about 3.5 to 5.5; phosphatebuffer (PBS) having pH 6 to 7.8, and citrate buffer having pH in therange of about 4 to
 6. 33. The process as claimed in claim 25, whereinsaid temperature is 37° C.
 34. The process as claimed in claim 28,wherein said temperature is 37° C.
 35. The process as claimed in claim28, wherein pH of said solution is 6.8 to 7.4 preferably 7.2.
 36. Theprocess as claimed in claim 25, wherein said period is 6-192 hours. 37.The process as claimed in claim 28, wherein pH of said solution is 6.8to 7.4 preferably 7.2.
 38. The process as claimed in claim 28, whereinsaid period is 6-192 hours.
 39. A method for treating metabolicdisorders selected from the group consisting of type 1, type 2 diabetesmellitus and complications thereof, wherein said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the pharmaceutical composition comprising the supramolecularinsulin assembly as claimed in claim 17 which is effective for thealleviation of said disorder.
 40. A method for treating metabolicdisorders selected from the group consisting of type 1, type 2 diabetesmellitus and complications thereof, wherein said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the pharmaceutical composition comprising the supramolecularinsulin assembly as claimed in claim 18, which is effective for thealleviation of said disorder.